Temperature-sensitive Transient Receptor Potential Research Articles

Brown Fat and Metabolic Health: The Diverse Functions of Dietary Components.

Brown and beige adipocytes utilize a variety of substrates for cold-induced thermogenesis, contributing to the clearance of metabolites in circulation and, consequently, metabolic health. Food-derived compounds that exhibit agonistic activity at temperature-sensitive transient receptor potential channels may serve as cold mimics to elicit thermogenesis and substrate utilization in brown adipose tissue (BAT). In addition to fatty acids and glucose, branched-chain amino acids (BCAAs), which are essential amino acids obtained from foods, are actively catabolized in BAT through mitochondrial BCAA carrier (MBC). The relative contribution of BCAAs to fueling the tricarboxylic acid cycle as a substrate (i.e., anaplerosis) is estimated to be relatively small, yet BCAA catabolism in BAT exerts a critical role in systemic insulin sensitivity. The nature of this apparent tension remained unclear until the recent discovery that active BCAA catabolism in BAT through MBC is critical for the synthesis of metabolites such as glutathione, which is delivered to the liver to improve hepatic insulin sensitivity through redox homeostasis. Novel mechanistic insights into the control of BAT function and systemic metabolism reveal the therapeutic potential of food-derived compounds for improving metabolic flexibility and insulin sensitivity.

Graphdiyne as a Highly Efficient and Neuron-Targeted Photothermal Transducer for in Vivo Neuromodulation.

Photothermal modulation of neural activity offers a promising approach for understanding brain circuits and developing therapies for neurological disorders. However, the low neuron selectivity and inefficient light-to-heat conversion of existing photothermal nanomaterials significantly limit their potential for neuromodulation. Here, we report that graphdiyne (GDY) can be developed into an efficient neuron-targeted photothermal transducer for in vivo modulation of neuronal activity through rational surface functionalization. We functionalize GDY with polyethylene glycol (PEG) through noncovalent hydrophobic interactions, followed by antibody conjugation to specifically target the temperature-sensitive transient receptor potential cation channel subfamily V member 1 (TRPV1) on the surface of neural cells. The nanotransducer not only exhibits high photothermal conversion efficiency in the near-infrared region but also shows great TRPV1-targeting capability. This enables photothermal activation of TRPV1, leading to neurotransmitter release in cells and modulation of neural firing in living mice. With its precision and selectivity, the GDY-based transducer provides an innovative avenue for understanding brain function and developing therapeutic strategies for neurodegenerative diseases.

Ingredients of Vicks VapoRub inhibit rhinovirus-induced ATP release.

Over-the-counter therapies, such as Vicks VapoRub, are frequently used in the management of upper respiratory tract infection symptoms. Of these, acute cough is the most bothersome; however, the mechanisms involved have not been fully elucidated. The temperature-sensitive transient receptor potential (TRP) channels, including TRPA1, TRPV1, TRPM8 and TRPV4, are potential candidates. TRPV4 is also thought to be involved in cough through the TRPV4-ATP-P2X3 pathway. Here, we hypothesise that Vicks VapoRub ingredients (VVRIs) modulate the TRP cough channels. Stably transfected HEK cells expressing TRP channels were challenged with VVRIs, individually or in combination, and the agonist and antagonist effects were measured using calcium signalling responses. In addition, rhinovirus serotype-16 (RV16)-infected A549 airway epithelial cells were pre-incubated with individual or combinations of VVRIs prior to hypotonic challenge and extracellular ATP release analysis. Calcium signalling reconfirmed some previously defined activation of TRP channels by specific VVRIs. The combined VVRIs containing menthol, camphor and eucalyptus oil activated TRPV1, TRPV4, TRPM8 and untransfected wild-type HEK293 cells. However, pre-incubation with VVRIs did not significantly inhibit any of the channels compared with the standard agonist responses. Pre-incubation of RV16-infected A549 cells with individual or combined VVRIs, except menthol, resulted in a 0.45-0.55-fold reduction in total ATP release following hypotonic stimulation, compared with infected cells not treated with VVRIs. These findings suggest that some VVRIs may reduce symptoms associated with upper respiratory tract infection by modulating specific TRP receptors and by reducing RV16-induced ATP release.

FRET analysis of the temperature-induced structural changes in human TRPV3

Transient receptor potential vanilloid member 3 (TRPV3) is an ion channel that plays a critical role in temperature sensing in skin. There have been active studies on how TRPV3, which is also known as one of the temperature-sensitive transient receptor potential (thermoTRP) channels, responds to temperature. However, the previous studies were mostly based on TRPV3 originating from mice or rats. Here, we focus on human TRPV3 (hTRPV3) and show that which domain of hTRPV3 undergoes conformational changes as temperature increases by Förster resonance energy transfer (FRET) assay. During the heat-induced activation of hTRPV3, the linker domain close to C-terminus, that is, the C-terminal domain shows a largest structural change whereas there is little change in the ankyrin repeat domain (ARD). Interestingly, the activation of hTRPV3 by an agonist shows structural change patterns that are completely different from those observed during activation by heat; we observe structural changes in ARD and S2–S3 linker after ligand stimulation whereas relatively little change is observed when stimulated by heat. Our results provide insight into the thermal activation of hTRPV3 channel.

Targeting temperature-sensitive transient receptor potential channels in hypertension: far beyond the perception of hot and cold.

Transient receptor potential (TRP) channels are nonselective cation channels and participate in various physiological roles. Thus, changes in TRP channel function or expression have been linked to several disorders. Among the many TRP channel subtypes, the TRP ankyrin type 1 (TRPA1), TRP melastatin type 8 (TRPM8), and TRP vanilloid type 1 (TRPV1) channels are temperature-sensitive and recognized as thermo-TRPs, which are expressed in the primary afferent nerve. Thermal stimuli are converted into neuronal activity. Several studies have described the expression of TRPA1, TRPM8, and TRPV1 in the cardiovascular system, where these channels can modulate physiological and pathological conditions, including hypertension. This review provides a complete understanding of the functional role of the opposing thermo-receptors TRPA1/TRPM8/TRPV1 in hypertension and a more comprehensive appreciation of TRPA1/TRPM8/TRPV1-dependent mechanisms involved in hypertension. These channels varied activation and inactivation have revealed a signaling pathway that may lead to innovative future treatment options for hypertension and correlated vascular diseases.

"ThermoTRP" Channel Expression in Cancers: Implications for Diagnosis and Prognosis (Practical Approach by a Pathologist).

Temperature-sensitive transient receptor potential (TRP) channels (so-called "thermoTRPs") are multifunctional signaling molecules with important roles in cell growth and differentiation. Several "thermoTRP" channels show altered expression in cancers, though it is unclear if this is a cause or consequence of the disease. Regardless of the underlying pathology, this altered expression may potentially be used for cancer diagnosis and prognostication. "ThermoTRP" expression may distinguish between benign and malignant lesions. For example, TRPV1 is expressed in benign gastric mucosa, but is absent in gastric adenocarcinoma. TRPV1 is also expressed both in normal urothelia and non-invasive papillary urothelial carcinoma, but no TRPV1 expression has been seen in invasive urothelial carcinoma. "ThermoTRP" expression can also be used to predict clinical outcomes. For instance, in prostate cancer, TRPM8 expression predicts aggressive behavior with early metastatic disease. Furthermore, TRPV1 expression can dissect a subset of pulmonary adenocarcinoma patients with bad prognosis and resistance to a number of commonly used chemotherapeutic agents. This review will explore the current state of this rapidly evolving field with special emphasis on immunostains that can already be added to the armoire of diagnostic pathologists.

Structural snapshots of the mechanism of TRPV2 channel activation by small-molecule agonists

Transient receptor potential (TRP) channels are polymodal sensors that play critical roles in various physiological processes in living organisms. These cation-permeable channels respond to a variety of physical and chemical stimuli, including cold and hot temperatures, acidic pH, and mechanical stress, often determining a sensory frontier of defense against hostile environments. Vanilloid (V) subfamily is the most studied category of TRP channels that includes six closely related members: highly calcium-selective TRPV5-6 and non-selective TRPV1-4. A remarkable feature of TRPV1-4 is their ability to sense heat, which makes them temperature-sensitive TRP channels or thermo-TRPs. TRPV channels are associated with a multitude of human diseases, including cancers, chronic pain, cardiovascular, neurological and nociceptive disorders. Despite the great clinical interest, pharmacology of TRPV channels remains largely undeveloped because of insufficient knowledge about the mechanisms of their regulation. For instance, activation of TRPV channels by small molecules or heat remains poorly understood. Numerous identified TRPV channel agonists, while effective in physiological experiments, appear limited in their ability to act in the conditions of structural biology experiments. In this regard, the recent study by Pumroy et al. [1] makes a significant contribution towards our understanding of TRPV2 structural dynamics that leads to opening of this channel in physiological conditions.

Coassembly of Warm Temperature-Sensitive Transient Receptor Potential Vanilloid (TRPV) 3 and TRPV4 Channel Complexes with Distinct Functional Properties.

Heteromeric assembly of temperature-sensitive transient receptor potential (TRP) ion channels has been suggested to underlie the molecular basis of fine-tuning of temperature detection and chemical sensation. However, whether warm temperature-sensitive TRP vanilloid (TRPV) 3 and TRPV4 channels robustly expressed in the skin can form heteromeric assembly remains largely unknown. In this study, we show that TRPV3 and TRPV4 channels can coassemble into functional heterotetrameric channels with distinct properties. Confocal imaging reveals a colocalization and association of TRPV3 and TRPV4 proteins in cell membrane. Coimmunoprecipitation analysis demonstrates a strong protein-protein interaction between TRPV3 and TRPV4 subunits from heterogeneously expressed cells or mouse skin tissues through their C termini but not in TRPV3 knockout tissues. Coexpression of TRPV3 and TRPV4 channels yields a heterotetrameric channel complexes characterized by an intermediate single-channel conductance, distinct activation threshold, and pharmacology. Taken together, our findings demonstrate a heterotetrameric assembly of TRPV3 and TRPV4 channels, which may help explain the role of temperature-sensitive TRPV channels in fine-tuning of environmental detection and sensation in the skin. SIGNIFICANCE STATEMENT: The coassembly of transient receptor potential vanilloid (TRPV) 3 and TRPV4 channel complexes increases the functional diversity within the channel subfamily, which may serve as a molecular basis for fine-tuning of environmental detection and temperature sensation in mammals.

Tether-free photothermal deep-brain stimulation in freely behaving mice via wide-field illumination in the near-infrared-II window.

Invasive brain implants and tethered optical fibres are typically used in restrained or motion-impaired animals, limiting the control and the decoding of the neural circuitry in freely behaving ones. Here we report the implant- and tether-free optical neurostimulation of deep brain regions by locally injected and untargeted photothermal transducers. The macromolecular transducers, comprising a semiconducting polymer core and an amphiphilic polymer shell, have an average diameter of 40 nanometres and achieve a photothermal conversion of 71% (at 1064 nm), activating the transient receptor potential cation channel subfamily V member 1 (TRPV1) ectopically expressed by an adeno-associated virus in dopaminergic neurons of tyrosine hydroxylase-driven Cre recombinase transgenic mice. The near-transparency of biological tissue in the second near-infrared window enabled the light source to be placed at 50 centimetres above the mouse, at a low incident power density of 10 milliwatt/square millimetre, resulting in the activation, through the scalp and skull, of the dopaminergic neurons in the ventral tegmental area, with minimal thermal damage. The approach is suitable for the neurostimulation of socially interacting mice.

Inhibition of temperature-sensitive TRPV3 channel by two natural isochlorogenic acid isomers for alleviation of dermatitis and chronic pruritus

Genetic gain-of-function mutations of warm temperature-sensitive transient receptor potential vanilloid 3 (TRPV3) channel cause Olmsted syndrome characterized by severe itching and keratoderma, indicating that pharmacological inhibition of TRPV3 may hold promise for therapy of chronic pruritus and skin diseases. However, currently available TRPV3 tool inhibitors are either nonselective or less potent, thus impeding the validation of TRPV3 as therapeutic target. Using whole-cell patch-clamp and single-channel recordings, we report the identification of two natural dicaffeoylquinic acid isomers isochlorogenic acid A (IAA) and isochlorogenic acid B (IAB) that selectively inhibit TRPV3 currents with IC50 values of 2.7 ± 1.3 and 0.9 ± 0.3 μmol/L, respectively, and reduce the channel open probability to 3.7 ± 1.2% and 3.2 ± 1.1% from 26.9 ± 5.5%, respectively. In vivo evaluation confirms that both IAA and IAB significantly reverse the ear swelling of dermatitis and chronic pruritus. Furthermore, the isomer IAB is able to rescue the keratinocyte death induced by TRPV3 agonist carvacrol. Molecular docking combined with site-directed mutations reveals two residues T636 and F666 critical for the binding of the two isomers. Taken together, our identification of isochlorogenic acids A and B that act as specific TRPV3 channel inhibitors and gating modifiers not only provides an essential pharmacological tool for further investigation of the channel pharmacology and pathology, but also holds developmental potential for treatment of dermatitis and chronic pruritus.

Structural mechanism of heat-induced opening of a temperature-sensitive TRP channel.

Numerous physiological functions rely on distinguishing temperature through temperature-sensitive transient receptor potential channels (thermo-TRPs). Although the function of thermo-TRPs has been studied extensively, structural determination of their heat- and cold-activated states has remained a challenge. Here, we present cryo-EM structures of the nanodisc-reconstituted wild-type mouse TRPV3 in three distinct conformations: closed, heat-activated sensitized and open states. The heat-induced transformations of TRPV3 are accompanied by changes in the secondary structure of the S2-S3 linker and the N and C termini and represent a conformational wave that links these parts of the protein to a lipid occupying the vanilloid binding site. State-dependent differences in the behavior of bound lipids suggest their active role in thermo-TRP temperature-dependent gating. Our structural data, supported by physiological recordings and molecular dynamics simulations, provide an insight for understanding the molecular mechanism of temperature sensing.

Temperature-sensitive transient receptor potential vanilloid channels: structural insights into ligand-dependent activation.

Temperature-sensitive transient receptor potential vanilloid ion channel subtypes 1-4 (thermoTRPV1-thermoTRPV4) play important roles in a wide range of physiological processes, including temperature sensing and body temperature regulation, inflammation, pain, itch, maintenance of skin, bone and hair, along with osmotic regulation. ThermoTRPV function is modulated by numerous natural product compounds, such as capsaicin, camphor and cannabinoids. Because of their physiological importance and their druggability, these channels have made attractive potential targets for drug development. Since the beginning of the cryo-electron miscroscopy (cryo-EM) "resolution revolution," a lot of progress has been made towards dissecting the activation mechanisms of thermoTRPVs and mapping the binding sites for modulatory compounds. Here, we review the thermoTRPV physiology and pharmacology and summarize the current knowledge of their mechanisms of gating and ligand binding sites. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

Ruthenium red attenuates brown adipose tissue thermogenesis in rats.

Ruthenium red (RR) is a non-selective antagonist of the temperature-sensitive Transient Receptor Potential (TRP) channels and it is an important pharmacological tool in thermoregulatory research. However, the effect of RR on thermoeffector activity is not well established. Here we evaluated the effect of RR on cold-defense thermoeffectors induced by menthol, an agonist of the cold-sensitive TRPM8 channel. Adult male Wistar rats were used. Epidermal treatment with menthol raised deep body temperature due to an increase in oxygen consumption (an index of thermogenesis), a reduction in heat loss index (an index of cutaneous vasoconstriction), and an induction in warmth-seeking behavior in a two-temperature choice apparatus. Pretreatment with RR attenuated the menthol-induced increase in deep body temperature and oxygen consumption, but it did not affect heat loss index and warmth-seeking behavior. To stimulate brown adipose tissue thermogenesis, rats were treated with CL 316,243, a potent and selective β3-adrenoceptor agonist. CL 316,243 increased deep body temperature, which was attenuated by RR pretreatment. We conclude that RR reduces brown adipose tissue thermogenesis induced by menthol and CL 316,243, independent of effects at the thermal sensor level (i.e., TRPM8).

TRPV4 Increases the Expression of Tight Junction Protein-Encoding Genes via XBP1 in Mammary Epithelial Cells.

Simple SummaryMammary glands are exocrine tissue, capable of secreting adequate amounts of milk protein during lactation. Each mammary gland is occupied by numerous alveoli. Each alveolus is composed of a single layer of mammary epithelial cells, adipose tissue, and ducts. Recent studies indicate that mild heat treatment of mammary epithelial cells at 39 °C has activated milk production. These results suggest that temperature may influence the physiological functions of mammary epithelial cells. In this study, we found that the temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) was involved in the increase of β-casein and TJ protein-encoding gene expression in response to mild heat treatment. On the other hand, severe heat treatment (41 °C) reduced the cell viability. Moreover, the Trpv4 mRNA level was significantly increased at Day 15 of gestation when the mammary alveoli are formed. TRPV4 is activated not only by temperature but also by mechanical forces that guide mammary epithelial development in the normal mammary gland. Our data suggest that TRPV4 has a possible function in mammary gland development.Mild heat stress (39 °C–40 °C) can positively regulate cell proliferation and differentiation. Indeed, mild heat treatment at 39 °C enhances the less-permeable tight junctions (TJs) formation and milk production in mammary epithelial cells. However, the molecular mechanisms of this response have not yet been delineated. In this study, the involvement of temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) in the increase of β-casein and TJ protein-encoding gene expression in response to mild heat treatment (39 °C) has been explored using HCll mouse mammary epithelial cells. Severe heat treatment (41 °C) induced the transcriptional level of Chop (C/EBP homologous protein; proapoptotic marker) and reduced the cell viability. It is speculated that the difference in unfolded protein response (UPR) gene expression upon stimulation at 39 °C vs. 41 °C controls cell survival vs. cell death. The accumulation of Trpv4 mRNA was significantly higher in 39 °C heat treatment cells. The β-casein, Zo-1 (zona occludens-1), Ocln (occludin), and Cldn3 (claudin 3) transcript levels were significantly increased in response to the addition of a selective TRPV4 channel agonist (GSK1016790A) at 37 °C. TRPV4 stimulation with GSK1016790A also increased the X-box-binding protein 1 splicing form (Xbp1s) at the transcript level. The increase in the mRNA levels of β-casein, Zo-1, Ocln, and Cldn3 in response to 39 °C heat treatment was suppressed by XBP1 knockdown. Moreover, the transcript level of Trpv4 was significantly increased at Day 15 of gestation, and its expression declined after 1 day of lactation. TRPV4 is activated not only by temperature but also by mechanical forces, such as cell stretching and shear stress, which guide mammary epithelial development in a normal mammary gland. These findings provide new insights of the possible function of TRPV4 in mammary gland development.

Biomimetic temperature-gated 2D cationic nanochannels for controllable osmotic power harvesting

Abstract In sensory neurons of mammals, some temperature-sensitive transient receptor potential (thermoTRP) ion channels open its pore for cations passing through by the activation of a high ambient temperature above the threshold. This thermal stimulus promotes the conversion from intracellular osmotic energy to the electrical signal, which inspires us to control energy output from nanochannel-based osmotic power harvesting systems. Here, temperature-gated 2D cationic nanochannels, stemming from the stacking of functionalized montmorillonite (MMT) lamellae, are constructed for controllable osmotic energy harvesting. Through electronegative modification, nanochannels demonstrate an excellent cation selectivity that is supported by both experimental and theoretical findings. When serving as a separator for osmotic power harvesting, cationic nanochannel membrane could deliver an output power of approximately 150 mW m−2, which is envisaged to be boosted by reducing the membrane resistance. Based on the temperature-gated performance of nanochannels, the energy output form osmotic power harvesting system could be regulated by alternating temperature switches in a reversible and stable manner. The output power on the external load resistance is doubled under a mild temperature rise from 30 °C to 60 °C. The strategy that combines intelligent response with osmotic power harvesting anticipates wide potentials for controllable energy utilizations.

Structure-Function Relationship of the Thermo-Sensitive TRP Channel TRP1 from the Alga ChlamydomonasReinhardtii

The guidance systems of algae to their habitat and environmental changes involve temperature-sensitive Transient Receptor Potential (TRP) ion channels, which are evolutionary predecessors of their mammalian orthologs. Here, we undertook structural and functional characterization of the representative TRP channel from alga Chlamydomonas reinhardtii, crTRP1. We determined a 3.5-Å resolution cryo-EM structure of crTRP1, which deviates significantly from the structures of other TRP channels and has a unique 2-fold symmetrical rose-shape architecture with elbow domains and ankyrin repeat domains submerged and dipping into the membrane, respectively. We found that the crTRP1 function is tightly regulated by phosphoinositides. No channel openings were observed when crTRP1 was incorporated into the bilayers in the absence of phosphoinositides, while the addition of phosphatidylinositol-4,5-bisphosphate (PIP2) or phosphatidylinositol-4-phosphate PI(4)P readily evoked channel activity. The crTRP1 channel demonstrated outwardly rectifying currents, where rectification resulted from both – differences in the single-channel conductance and open probability (Po). Strikingly, crTRP1 exhibited high temperature sensitivity, similar to the mammalian temperature-sensitive TRP channels (thermoTRPs). The temperature dependence of the crTRP1 channel openings yielded a high value of the temperature coefficient, Q10 = 25 ± 1.17, that closely resembles the Q10 for the mammalian thermoTRPs. Combining the first structure of a TRP channel from a microorganism with its functional characterization, we provide a framework for better understanding of alga biology and TRP channel evolution.

Regulatory switch at the cytoplasmic interface controls TRPV channel gating.

Temperature-sensitive transient receptor potential vanilloid (thermoTRPV) channels are activated by ligands and heat, and are involved in various physiological processes. ThermoTRPV channels possess a large cytoplasmic ring consisting of N-terminal ankyrin repeat domains (ARD) and C-terminal domains (CTD). The cytoplasmic inter-protomer interface is unique and consists of a CTD coiled around a β-sheet which makes contacts with the neighboring ARD. Despite much existing evidence that the cytoplasmic ring is important for thermoTRPV function, the mechanism by which this unique structure is involved in thermoTRPV gating has not been clear. Here, we present cryo-EM and electrophysiological studies which demonstrate that TRPV3 gating involves large rearrangements at the cytoplasmic inter-protomer interface and that this motion triggers coupling between cytoplasmic and transmembrane domains, priming the channel for opening. Furthermore, our studies unveil the role of this interface in the distinct biophysical and physiological properties of individual thermoTRPV subtypes.

Bionic Thermoelectric Response with Nanochannels.

Thermosensation, the ability to detect environmental temperature change, is one of most ancient and crucial processes for the survival of all living organisms. Mammals use temperature-sensitive transient receptor potential (thermoTRP) cation channels as thermometers to convert the temperature change into electrical signals that are finally received as action potentials by nerve endings. In this work, we report the bionic thermosensation by solid-state hybrid nanochannels based on the principle of thermally sensitive permselective ion transport. The hybrid nanochannels possess an asymmetric structure, consisting of ultrasmall silica nanochannels (∼2.3 nm in diameter, ∼100 nm in length) supported by large-sized track-etched poly(ethylene terephthalate) conical nanochannels. When the hybrid nanochannels are engineered to separate two electrolyte solutions, the temperature change in one solution can be directly converted into trans-nanochannel diffusion potential, akin to the natural thermosensation process. Two bionic modes, namely, in the absence and presence of a concentration gradient, were studied to imitate the natural thermosensation of thermoTRP ion channels and shark, respectively. In both cases, real-time thermoelectric response was captured with a fast relative response speed (electrical response time versus temperature change time) of higher than 98%, as well as excellent stability and reversibility. Moreover, the nanochannels are highly sensitive to thermal stimulus, showing a sensitivity of 0.71 mV/K comparable to the natural thermosensation. The experimental results coincide well with the theoretical relationship between electrical response and temperature change derived in terms of a quasi-steady-state ion transport model. Finite element simulations based on coupled Poisson-Nernst-Planck (PNP) and Einstein-Stokes equations were also performed, confirming that the sensitive thermoelectric response originates from the highly cationic selectivity of nanochannels.

Mechanism for Regulation of Melanoma Cell Death via Activation of Thermo-TRPV4 and TRPV2.

Background Thermo-TRPs (temperature-sensitive transient receptor potential channels) belong to the TRP (transient receptor potential) channel superfamily. Emerging evidence implied that thermo-TRPs have been involved in regulation of cell fate in certain tumors. However, their distribution profiles and roles in melanoma remain incompletely understood. Methods Western blot and digital PCR approaches were performed to identify the distribution profiles of six thermo-TRPs. MTT assessment was employed to detect cell viability. Flow cytometry was applied to test cell cycle and apoptosis. Calcium imaging was used to determine the function of channels. Five cell lines, including one normal human primary epidermal melanocytes and two human malignant melanoma (A375, G361) and two human metastatic melanoma (A2058, SK-MEL-3) cell lines, were chosen for this research. Results In the present study, six thermo-TRPs including TRPV1/2/3/4, TRPA1, and TRPM8 were examined in human primary melanocytes and melanoma cells. We found that TRPV2/4, TRPA1, and TRPM8 exhibited ectopic distribution both in melanocytes and melanoma cells. Moreover, activation of TRPV2 and TRPV4 could lead to the decline of cell viability for melanoma A2058 and A375 cells. Subsequently, activation of TRPV2 by 2-APB (IC50 = 150 μM) induced cell necrosis in A2058 cells, while activation of TRPV4 by GSK1016790A (IC50 = 10 nM) enhanced apoptosis of A375 cells. Furthermore, TRPV4 mediated cell apoptosis of melanoma via phosphorylation of AKT and was involved in calcium regulation. Conclusion Overall, our studies revealed that TRPV4 and TRPV2 mediated melanoma cell death via channel activation and characterized the mechanism of functional TRPV4 ion channel in regulating AKT pathway driven antitumor process. Thus, they may serve as potential biomarkers for the prognosis and are targeted for the therapeutic use in human melanoma.

Effect of Danggui Sini Decoction on the Behaviour and Dorsal Root Ganglion TRP Channel of Neuropathic Pain in CCI Rats

Danggui Sini decoction, a traditional Chinese medicine formula, has been extensively used as a remedy for neuropathic pain and other diseases in recent years. To explore whether Danggui Sini decoction can alleviate mechanical pain, thermal hyperalgesia, and cold pain at the same time, the role of temperature-sensitive transient receptor potential channel in neuropathic pain and the therapeutic effect of Danggui Sini decoction on neuropathic pain as well as the impact on the transient receptor potential pathway were explored in the present study. One hundred and twentyeight Sprague Dawley rats were separated randomly into 4 groups; the sham-operated group, chronic constriction injury of sciatic nerve model group, pregabalin group, and Danggui Sini decoction-treated group. The rats in the sham-operated group underwent surgery, but no nerve was ligated. The rest of the rats were subjected to sciatic nerve ligation. Rats in Danggui Sini decoction-treated group could be irrigated to Danggui Sini decoction. Rats in the sham-operated group and chronic constriction injury model group were gavaged with saline. Rats in pregabalin group were given pregabalin. Behavioral tests conducted were, acetone test and cold plate, hot plate experiment, and von Frey filaments on 4, 7, 14, 21st day, and then dorsal root ganglion neurons were take out. Nerve ligation from the ipsilateral L4-6 dorsal root ganglion were measured TRPM8, TRPA1, TRPV1, TRPV2, TRPV3, and TRPV4 proteins expression by immunofluorescence staining and Western-blot method. Danggui Sini decoction could significantly inhibit the cold allodynia and the mechanical allodynia. The immunofluorescence studies showed that the model group of dorsal root ganglia on TRPM8, TRPA1, TRPV1, TRPV2, TRPV3, and TRPV4 proteins were decreased. Western-blot results showed the the expression of TRPM8, TRPA1, TRPV1, TRPV2, TRPV3, and TRPV4 proteins in Danggui Sini decoction-treated group was reduced. Danggui Sini decoction could not only suppress the cold allodynia, but also alleviate mechanical allodynia and thermal hyperalgesia. Danggui Sini decoction could down-regulate abnormally high expression of temperature-dependent transient receptor potential channel protein.