Potential Channels Research Articles

Functional characterizations of thermosensitive TRPV channels from Holocephalan elephant shark, Callorhinchus milii, illuminate the ancestral thermosensory system in vertebrates.

Homeostasis and survival of various animal species have been affected by changes in environmental temperature, causing animals to evolve physiological systems for sensing ambient and body temperature. Temperature-sensitive transient receptor potential (TRP) channels have multimodal properties that are activated by physical stimuli such as temperature, as well as by various chemical substances. Our goal is to understand the diversity of the vertebrate thermosensory system by characterizing the temperature-sensitive TRPV channels of the elephant shark, which belongs to the holocephali of the cartilaginous fishes. Since elephant sharks are basal jawed vertebrates, analysis of elephant shark TRPs is critical to understanding the evolution of thermosensory systems in vertebrate lineages. We found that temperature stimulation activated elephant shark TRPVs in an electrophysiological analysis similar to the mammalian orthologue. The thermal activation threshold of elephant shark TRPV1 (31°C) was similar to the thresholds reported for several other fish species, but was much lower than that of mammalian orthologs. Strikingly, the elephant shark TRPV4 was a cooling-activated channel with a threshold of 20°C, whereas, in several tetrapods, it is activated by warmth. These results suggest that the temperature sensitivity of TRPV4 has changed in vertebrate evolutionary lineages. Furthermore, we also found the elephant shark possesses heat-evoked TRPV3, which has a threshold of 42°C, which is absent in more derived teleost fishes. Taken together, our findings elucidate that the vertebrate-type thermosensory system has already emerged in the common ancestor of jawed vertebrates, although their temperature sensing ranges were different from those of mammals.

CaBind_MCNN: Identifying Potential Calcium Channel Blocker Targets by Predicting Calcium-Binding Sites in Ion Channels and Ion Transporters Using Protein Language Models and Multiscale Feature Extraction.

Calcium ions (Ca2+) are crucial for various physiological processes, including neurotransmission and cardiac function. Dysregulation of Ca2+ homeostasis can lead to serious health conditions such as cardiac arrhythmias and hypertension. Ion channels and transporters play a vital role in maintaining cellular Ca2+ balance by facilitating Ca2+ transport across cell membranes. Accurate prediction of Ca2+ binding sites within these proteins is essential for understanding their function and identifying potential therapeutic targets, particularly for developing novel calcium channel blockers (CCBs). This study introduces CaBind_MCNN, an innovative computational model that leverages pretrained protein language models (PLMs) and a multiscale feature extraction approach to predict Ca2+ binding sites in ion channels and transporter proteins. Our method integrates embeddings from the ProtTrans PLM with a convolutional neural network (CNN)-based multiwindow scanning approach, capable of capturing diverse sequence features relevant to Ca2+ binding. The model, trained on a curated data set of 27 calcium-binding protein sequences, achieves high accuracy with an area under the curve (AUC) of 0.9886, significantly outperforming some existing methods. These results demonstrate the potential of CaBind_MCNN to enhance drug discovery efforts by identifying potential CCB targets and advancing the development of novel therapies for calcium-related disorders.

Loss of Endothelial TRPC1 Induces Aortic Hypercontractility and Hypertension.

The increasing prevalence of obesity-related cardiovascular diseases demands a better understanding of the contribution of different cell types to vascular function for developing new treatment strategies. Previous studies have established a fundamental role of TRPC1 (transient receptor potential channel canonical family member 1) in blood vessels. However, little is known about its functional roles within different cell types. We generated endothelial-specific TRPC1-deficient and knockin mice and analyzed their changes in vascular function under physiological and pathologically obese state. Wire myography, Ca2+ image, blood pressure measurements, RNA-sequencing analysis, liquid chromatography-mass spectrometry, immunoblotting, ELISA, luciferase reporter assay, and morphometric assessments were performed to unravel phenotype and molecular changes in response to the absence or presence of endothelial TRPC1. Loss of endothelial TRPC1 reduced endothelial-dependent relaxation and exaggerated endothelial-dependent contraction in mouse aorta. As expected, loss of endothelial TRPC1 amplified blood pressure and decreased acetylcholine-induced intracellular Ca2+ concentration rise in the aorta. In endothelial-specific TRPC1-deficient mouse arteries, the mRNA profile identified upregulation of c-Fos. Blockade of c-Fos rescued the impaired vasomotor tone in the aorta of mice deficient in endothelial TRPC1. Endothelial TRPC1-regulated nitric oxide/endothelin-1 production is involved in vascular c-Fos expression. Moreover, knockin of endothelial TRPC1 ameliorated enhanced endothelial-dependent contraction and hypertension in obese mice which is related to alleviated endothelial endothelin-1/c-Fos production and smooth muscle contraction. Our results identify endothelial TRPC1 as a previously unclear regulator of vascular changes and blood pressure in both physiological and pathologically obese state, and it is associated with nitric oxide/endothelin-1/c-Fos signaling.

Hospital supply and infant mortality: Evidence from Islamic political representation in Turkey

We investigate the impact of healthcare facility supply on infant mortality by exploiting a change in local governments in Turkey as a quasi-exogenous shock to local hospital supply. Using a regression discontinuity design, we find a significant decline in infant mortality in municipalities where an Islamic mayor was barely elected. Exploring potential channels, we document a significant increase in the number and size of hospitals and health clinics in treated municipalities. Moreover, children in these municipalities are more likely to be born in government facilities, receive treatment for cough and fever, benefit from a greater number of childhood vaccinations, and have more prenatal care visits before birth. Our findings highlight that investments in public healthcare facilities in disadvantaged areas can have substantial effects on the survival of infants.

Microglia TRPC1 SUMOylation drives neuroinflammation after stroke by modulating NLRP3 activity via increasing TRPC1 interaction with ARRB2.

Microglial canonical transient receptor potential channel 1 (TRPC1) has been proposed to influence neuroinflammation after cerebral ischemia and reperfusion injury (CIRI), however, the underlying mechanism remains poorly understood. This study demonstrates that TRPC1 is modified by small ubiquitin-related modifier (SUMO)ylation. Our findings suggest a notable increase in microglial TRPC1 SUMOylation within both the middle cerebral artery occlusion reperfusion (MCAO/R) model and the in vitro oxygen-glucose deprivation/regeneration model. Mice with a loss of TRPC1 SUMOylation in microglia exhibited improved stroke outcomes including reduced behavior deficits, infarct volume, blood brain barrier damage as well as neuronal apoptosis. Mechanistically, SUMOylation of microglial TRPC1 exacerbated neutrophil infiltration into the peri-infarct area. Additionally, SUMOylated TRPC1 activates the Nod-like receptor protein (NLRP) 3 signaling pathway in microglia and stimulates multiple CC-chemokine ligands and C-X-C motif ligand chemokines after MCAO/R. SUMOylated TRPC1 facilitates the interaction between TRPC1 and β-arrestin2 (ARRB2), a negative regulator of NLRP3 inflammasome, which disrupts the NLPR3/ARRB2 complex and stimulates the activation of the NLPR3 signaling pathway. Furthermore, ARRB2 directly binds to the residues 46 to 61 of TRPC1 N terminus, which is enhanced by TRPC1 SUMOylation. Collectively, our findings demonstrate a previously unidentified mechanism by which SUMOylated TRPC1 in microglia regulates leukocyte infiltration after stroke, suggesting that the inhibition of microglial TRPC1 SUMOylation may provide therapeutic benefits for CIRI.

Mechanisms of chronic postsurgical pain

Chronic pain after surgery, also known as chronic postsurgical pain (CPSP), is recognized as a significant public health issue with serious medical and economic consequences. Current research on CPSP underscores...

Evaluation of immunohistochemical TRPC3 and TRPC6 expression patterns in human endometriosis.

Although to date the pathogenesis of endometriosis remains largely unexplained, it is known that processes of migration, proliferation and revascularization and thus calcium as a messenger substance play an important role. Consecutively, the present study examines the immunohistochemical expression of the calcium transient receptor potential channels 3 and 6 (TRPC3 and TRPC6) in ectopically located (outside the uterine cavity) endometrial tissue. Laparoscopically collected and histomorphologically verified endometriosis tissues from several different intraabdominal locations were examined (n = 20) and immunohistochemical stainings were performed with anti-TRPC3 and anti-TRPC6 antibodies (Alomone Labs, Jerusalem). Hereby, eutopic endometrium served as a healthy control cohort (n = 6). Staining patterns were evaluated using a modified immunoreactive score (IRS) and exploratory statistical analysis was performed, aiming to determine relations of staining intensities with associated clinical parameters such as rASRM stage and location. We determined a strong cytoplasmatic TRPC3 and TRPC6 expression in all ectopic endometrial glandular formations, albeit with focally varying staining intensities. Within our cohort, we did not verify a statistically significant difference of TRPC3 and TRPC6 expression between endometriosis patients and a healthy control group or between different clinical affections (rASRM stages). Our study confirms - to our knowledge for the first time - the successful immunohistochemical assessment of TRPC3 and TRPC6 in endometriosis, setting the basis for future studies aiming at evaluating not only clinical aspects of TRPC3 and TRPC6 expression but also shedding light on its function in the pathophysiological context of endometriosis.

Neurotoxins Acting on TRPV1—Building a Molecular Template for the Study of Pain and Thermal Dysfunctions

Transient Receptor Potential (TRP) channels are ubiquitous proteins involved in a wide range of physiological functions. Some of them are expressed in nociceptors and play a major role in the transduction of painful stimuli of mechanical, thermal, or chemical origin. They have been described in both human and rodent systems. Among them, TRPV1 is a polymodal channel permeable to cations, with a highly conserved sequence throughout species and a homotetrameric structure. It is sensitive to temperature above 43 °C and to pH below 6 and involved in various functions such as thermoregulation, metabolism, and inflammatory pain. Several TRPV1 mutations have been associated with human channelopathies related to pain sensitivity or thermoregulation. TRPV1 is expressed in a large part of the peripheral and central nervous system, most notably in sensory C and Aδ fibers innervating the skin and internal organs. In this review, we discuss how the transduction of nociceptive messages is activated or impaired by natural compounds and peptides targeting TRPV1. From a pharmacological point of view, capsaicin—the spicy ingredient of chilli pepper—was the first agonist described to activate TRPV1, followed by numerous other natural molecules such as neurotoxins present in plants, microorganisms, and venomous animals. Paralleling their adaptive protective benefit and allowing venomous species to cause acute pain to repel or neutralize opponents, these toxins are very useful for characterizing sensory functions. They also provide crucial tools for understanding TRPV1 functions from a structural and pharmacological point of view as this channel has emerged as a potential therapeutic target in pain management. Therefore, the pharmacological characterization of TRPV1 using natural toxins is of key importance in the field of pain physiology and thermal regulation.

Inhibition of TRPM7 by glutathione decreases oxidant and apoptotic action of cisplatin through the downregulation of Ca2+ and Zn2+ in glioblastoma cells.

Cisplatin (CiSP)-mediated stimulation of TRPM7 may induce oxidant and apoptotic activities through the upregulation of Ca2+, apoptosis, and reactive oxygen species (ROS) in glioblastoma (DBTRG-05MG) cells, whereas inhibition of TRPM7 by the antioxidant glutathione (GSH) may reduce the observed increases in DBTRG-05MG. The aim of the study was to examine how TRPM7 activation stimulates DBTRG-05MG cell death but also how it inhibits the effects of TRPM7 antagonists (GSH and carvacrol, CRV) via altering ROS toxicity and apoptosis. In the DBTRG-05MG, 5 groups were established: control, GSH (10 mM for 2h), CiSP (25 μM for 24h), CiSP+GSH, and CiSP+CRV (200 mM for 24h). The amounts of cytosolic free Ca2+ were further increased in the CiSP group by the stimulation of TRPM7 (naltriben), even though the GSH and CRV treatments caused them to decrease in the cells. The amounts of mitochondrial membrane hyperpolarization, ROS, death cell, apoptosis, free zinc ion, and caspase-3, -8, and -9 in the cells were higher in the CiSP than in the control and GSH, although their amounts were lower in the CiSP+GSH and CiSP+CRV than in the CiSP only. The CiSP-induced decreases in cell viability and GSH concentrations were increased by GSH incubation. The stimulation of TRPM7 increased the anticancer action of CiSP, although its inhibition decreased the amount of CiSP-induced oxidative stress and DBTRG-05MG deaths through the treatment of GSH and CRV. TRPM7 stimulation could be considered a potential tumor killer channel through oxidative glioblastoma damage caused by CiSP.

A Practical and Highly Efficient Procedure for the Synthesis of the Trifluoromethylpyrazol-Derived Canonical Transient Receptor ­Potential Channel (TRPC3) Inhibitor Pyr3 and Its Derivatives

AbstractCanonical transient receptor potential (TRPC) channels play a variety of diverse biological functions. Among the subgroups of the TRPC family (TRPC3, TRPC6, and TRPC7), TRPC3 has been implicated in several diseases, including neurological and cardiovascular diseases, cancer, and neurodegeneration. A pyrazole-based compound Pyr3 is a selective TRPC3 channel inhibitor which is now a standard tool compound to study conditions associated with TRPC3 dysregulation. However, the reported literature syntheses of Pyr3 either provided low yield or involved microwave reactions and harsh reaction conditions. We have developed a practical and highly efficient procedure for the synthesis of Pyr3 and its derivatives in high yields which is suitable for scale-up synthesis.

Neuron Modulation by Synergetic Management of Redox Status and Oxidative Stress.

The transient receptor potential (TRP) channel is a key sensor for diverse cellular stimuli, regulating the excitability of primary nociceptive neurons. Sensitization of the TRP channel can heighten pain sensitivity to innocuous or mildly noxious stimuli. Here, reversible modulation of TRP channels is achieved by controlling both the light-induced photoelectrochemical reaction to induce neuronal depolarization, and antioxidants for neuronal protection. It is based on a hybrid nanosystem, CZPN, created by coating CeO2 nanocrystals with the metalloporphyrin ZnTPyP. Light irradiation triggers an electrochemical response, with efficient electron injection from ZnTPyP to CeO2, converting Ce4+ into Ce3+ as antioxidants. Meanwhile, the charge migrates from surrounding O2 molecules to the hole-injected ZnTPyP*, giving rise to reactive oxygen species (ROS). This change in the redox environment sensitizes TRP channels, eliciting action potentials in primary rat neurons, and is partially blocked by pretreatment with capsazepine. The resulting CeO2-x, with a high Ce3+/Ce4+ ratio, can scavenge excessive ROS to prevent oxidative damage. The light-induced pain behaviors in mice pre-injected with CZPN are further confirmed. This work suggests a safe, effective, and universal approach to photoelectrochemical processes for modulation and research of the peripheral nervous system.

Noninterference Analysis of Reversible Systems: An Approach Based on Branching Bisimilarity

The theory of noninterference supports the analysis of information leakage and the execution of secure computations in multi-level security systems. Classical equivalence-based approaches to noninterference mainly rely on weak bisimulation semantics. We show that this approach is not sufficient to identify potential covert channels in the presence of reversible computations. As illustrated via a database management system example, the activation of backward computations may trigger information flows that are not observable when proceeding in the standard forward direction. To capture the effects of back-and-forth computations, it is necessary to switch to a more expressive semantics, which has been proven to be branching bisimilarity in a previous work by De Nicola, Montanari, and Vaandrager. In this paper we investigate a taxonomy of noninterference properties based on branching bisimilarity along with their preservation and compositionality features, then we compare it with the taxonomy of Focardi and Gorrieri based on weak bisimilarity.

First-principles study of anisotropic planar 2D BC2N for sub-5 nm high-performance p-type transistors.

Two-dimensional (2D) materials are considered the potential channel for next-generation transistors. Unfortunately, the development of p-type 2D material transistors lags significantly behind that of n-type, thereby impeding the advancement of complementary logical circuits. In this study, we investigated the electronic properties of 2D BC2N and analyzed the transport performance of p-type 2D BC2N-6 FETs through first-principles calculations. The anisotropic electronic properties of BC2N-6 led to variations in device transport performance along the zigzag and armchair directions. The on-state current of 10 nm BC2N-6 FETs could reach 2415 μA μm-1 and 1660 μA μm-1 along the zigzag and armchair directions, respectively. Subthreshold swing (SS) values for both directions were 63 mV dec-1, nearing the limit of 60 mV dec-1. Even when the gate length was scaled down to 5 nm, the on-state current of BC2N-6 FETs in both directions exceeded 1500 μA μm-1, which was approximately 160% of International Technology Roadmap for Semiconductors (ITRS) standards for high-performance (HP) devices. Furthermore, the delay time (τ) and power dissipation (PDP) of BC2N-6 FETs could fully satisfy ITRS requirements. Our work demonstrates that monolayer BC2N-6 can serve as a competitive p-type channel for next-generation devices.

Role of neurogenic inflammation in intervertebral disc degeneration.

In healthy intervertebral discs (IVDs), nerves and blood vessels are present only in the outer annulus fibrosus, while in degenerative IVDs, a large amount of nerve and blood vessel tissue grows inward. Evidence supports that neurogenic inflammation produced by neuropeptides such as substance P and calcitonin gene related peptide released by the nociceptive nerve fibers innervating the IVDs plays a crucial role in the process of IVD degeneration. Recently, non-neuronal cells, including IVD cells and infiltrating immune cells, have emerged as important players in neurogenic inflammation. IVD cells and infiltrating immune cells express functional receptors for neuropeptides through which they receive signals from the nervous system. In return, IVD cells and immune cells produce neuropeptides and nerve growth factor, which stimulate nerve fibers. This communication generates a positive bidirectional feedback loop that can enhance the inflammatory response of the IVD. Recently emerging transient receptor potential channels have been recognized as contributors to neurogenic inflammation in the degenerative IVDs. These findings suggest that neurogenic inflammation involves complex pathophysiological interactions between sensory nerves and multiple cell types in the degenerative IVDs. Clarifying the mechanism of neurogenic inflammation in IVD degeneration may provide in-depth understanding of the pathology of discogenic low back pain.

The Impact of Green Finance on Enterprise Environmental Strategies: Source Prevention or End‐Of‐Pipe Treatment?

ABSTRACTBased on the quasinatural experiment of China's green finance reform and innovation pilot zones (GFRIs), the impact of GFRIs on the environmental strategies of heavy‐polluting enterprises is discussed. The results show that after the implementation of GFRIs, heavy‐polluting enterprises are more inclined to end‐of‐pipe treatment than source prevention. Alleviating financing restrictions and enhancing external attention are potential channels for GFRIs to induce enterprises to adopt end‐of‐pipe treatment. Additionally, we explore the governance boundaries of GFRIs for heterogeneous characteristics of enterprises and the market environment. The findings enrich the research related to the economic consequences of GFRIs and provide empirical evidence to formulate the next step of pilot expansion.

Piezo Ion Channels and Their Association With Haptic Technology Use: A Narrative Review.

The recent identification of Piezo ion channels demonstrating a mechano-sensitive impact on neurons revealed distinct Piezo-1 and 2 types. While Piezo-1 predominates in neurons linked to non-sensory stimulation, such as pressure in blood vessels, Piezo-2 predominates in neurons linked to sensory stimulation, such as touch. Piezo-1 and 2 have a major bidirectional impact on transient receptor potential (TRP) ion channels, and TRPs also impact neurotransmitter release. Particularly existent in dorsal root ganglion (DRG) neurons, which are located in nerve endings, Piezo-2 is a key DRG activator. Subsequent Piezo findings have been vital to recent medical haptic technology developments, in tandem with breakthroughs in the emerging neurology subfield of connectomics plus AI developments. Included in this review are a historical Piezo overview, the interrelationship of Piezo channels with TRPs, inclusive of TRPV1/TRPV8, the impact on medical and rehab haptic technology, a focus on haptic technology use in stroke survivor rehab inclusive of pain mitigation, and the development of a haptic technology patch aimed at alleviating pain and/or anxiety. Neurogenic pain resulting from hyperalgesia/allodynia in stroke survivors is a potential target for drugs and haptics aimed at pain reduction; patients experiencing neuropathic or psychosomatic pain are other prime targets. Increased Piezo knowledge may promote more precisely targeted haptic therapeutic developments.

Molecular basis of neurosteroid and anticonvulsant regulation of TRPM3.

Transient receptor potential channel subfamily M member 3 (TRPM3) is a Ca2+-permeable cation channel activated by the neurosteroid pregnenolone sulfate (PregS) or heat, serving as a nociceptor in the peripheral sensory system. Recent discoveries of autosomal dominant neurodevelopmental disorders caused by gain-of-function mutations in TRPM3 highlight its role in the central nervous system. Notably, the TRPM3 inhibitor primidone, an anticonvulsant, has proven effective in treating patients with TRPM3-linked neurological disorders and in mouse models of thermal nociception. However, our understanding of neurosteroids, inhibitors and disease mutations on TRPM3 is limited. Here we present cryogenic electron microscopy structures of the mouse TRPM3 in complex with cholesteryl hemisuccinate, primidone and PregS with the synthetic agonist CIM 0216. Our studies identify the binding sites for the neurosteroid, synthetic agonist and inhibitor and offer insights into their effects and disease mutations on TRPM3 gating, aiding future drug development.

Marine-derived bioactive compounds for neuropathic pain: pharmacology and therapeutic potential.

Neuropathic pain, a challenging condition often associated with diabetes, trauma, or chemotherapy, impairs patients' quality of life. Current treatments often provide inconsistent relief and notable adverse effects, highlighting the urgent need for safer and more effective alternatives. This review investigates marine-derived bioactive compounds as potential novel therapies for neuropathic pain management. Marine organisms, including fungi, algae, cone snails, sponges, soft corals, tunicates, and fish, produce a diverse range of secondary metabolites with significant pharmacological properties. These include peptides (e.g., conopeptides, piscidin 1), non-peptides (e.g., guanidinium toxins, astaxanthin, docosahexaenoic acid, fucoidan, apigenin, fumagillin, aaptamine, flexibilide, excavatolide B, capnellenes, austrasulfones, lemnalol), and crude extracts (e.g., Spirulina platensis, Dunaliella salina, Cliothosa aurivilli). These compounds exhibit diverse mechanisms of action, such as modulating ion channels (e.g., transient receptor potential channels, voltage-gated sodium, calcium, and potassium channels, and G protein-coupled inwardly rectifying potassium channels), interacting with cell-surface receptors (e.g., nicotinic acetylcholine, NMDA, kainate, GABAB​, and neurotensin receptors), inhibiting norepinephrine transporters, reducing oxidative stress, and attenuating neuroinflammation. These effects collectively contribute to alleviating nerve degeneration and symptoms of neuropathic pain, including hyperalgesia, allodynia, and associated psychomotor disturbances. Marine-derived bioactive compounds represent promising alternatives to conventional neuropathic pain treatments, to advance their development and assess their integration into neuropathic pain management strategies.

Using electrical resistivity techniques (ERT and SP) for nondestructive detection of seepage channels at the Leitai heritage site, China

Seepage accelerates the weathering and destruction of cultural heritage sites, posing a major preservation challenge, while the concealed nature of seepage channels complicates their detection due to noninvasive requirements. In this study, we applied a comprehensive geophysical approach, integrating electrical resistivity tomography (ERT) and self-potential (SP) techniques, to image seepage channels within the Leitai heritage site. These potential seepage channels have already caused a collapse pit measuring 3.1 m × 2.7 m on the site’s surface. We began with 2D ERT surveys, which were then combined for 3D inversion to reveal the resistivity structure of the site. Subsequently, SP data were extracted along typical survey lines using interpolation algorithms, and these were inverted to supplement and verify the resistivity structure. The results from both techniques were highly consistent, indicating the presence of internal channels within the site. This comprehensive geophysical approach provides critical insights and references for the subsequent restoration efforts of the Leitai heritage site, ensuring the protection and preservation of this culturally significant landmark. Moreover, the method proposed in this study can be easily applied to the preservation of similar cultural heritage sites elsewhere.

A Study on the Regulation of TRPM8 Signaling Pathway by Interior-warming Medicines in Traditional Chinese Medicine

Background: Interior-warming medicines (Wēnlǐ herbs) have the effect of warming the interior of the body and dispelling cold, and they are traditionally used for interior cold syndrome. Currently, they are used for treating gastrointestinal disorders, rheumatoid arthritis, tumors, etc. Transient receptor potential channel M8 (TRPM8) is a non-selective cation channel that can be activated by low temperature and menthol. However, the mechanism of temperature regulation of interior- warming medicines is not clear. This study designed an in vitro experiment on the TRPM8 signaling pathway using interior-warming medicines, aiming to investigate the relationship between the hot property of interior-warming medicines and the regulatory effect of TRPM8. Methods: The breast cancer cell line 7 was cultured at different temperatures as a research model, six kinds of interior-warming medicines were used, and medicated serums from rats were prepared as test drugs. PCR and western blotting were carried out to investigate their effects on TRPM8 mRNA and protein expression. ELISA and flow cytometry were conducted to detect intracellular cAMP and Ca2+ concentration. Results: Compared with the blank-containing serum group, interior-warming medicines had no significant effect on the expression of TRPM8 at 37°C, while they could inhibit the expression of TRPM8 at low temperatures (30°C). Moreover, the six herbs could increase intracellular cAMP content and reduce Ca2+ concentration at different temperatures. Conclusion: The natural hot property of interior-warming medicines may be associated with the regulation of the TRPM8 signaling pathway, causing Ca2+ influx blocking, which provides a deeper experimental basis for their clinical application and new drug development.