Phospholipid asymmetry is a hallmark of mammalian cell membranes and reflects the selective distribution of distinct phospholipid species between the two leaflets of the lipid bilayer. Although this asymmetry is tightly maintained, the membrane proteins whose functions depend on it remain largely unknown. To perturb phospholipid asymmetry experimentally, we expressed a constitutively active phospholipid scramblase and thereby identified transient receptor potential melastatin 8 (TRPM8) as an ion channel regulated by this membrane property. Activation of TRPM8 by both l -menthol and innocuous cold was markedly suppressed following disruption of phospholipid asymmetry. Likewise, selective depletion of phosphatidylserine (PS), a phospholipid enriched in the cytoplasmic leaflet, using a cytosolically targeted PS decarboxylase attenuated TRPM8 activation, indicating that cytoplasmic PS is required for proper TRPM8 function. Mechanistically, our findings suggest that cytoplasmic PS supports efficient TRPM8 activation by maintaining the biochemical state of the channel. Together, these findings identify TRPM8 as a phospholipid asymmetry-dependent ion channel and establish an experimental strategy for systematically identifying membrane proteins regulated by phospholipid asymmetry. This work provides a foundation for future studies investigating the biological significance of this fundamental membrane property.

Most transient receptor potential (TRP) channels are Ca2+-permeable non-selective cation channels that function as polymodal receptors activated by a wide variety of stimuli, including natural compounds such as pungent substances, physical stimuli, lipids, intracellular signaling molecules, and ions. Their physiological roles are diverse, including sensory perception, ion transport, and intracellular signaling. Similarly, Piezo channels, which are also Ca2+-permeable non-selective cation channels, are activated by mechanical stimuli such as membrane stretching and contribute to touch sensation, blood flow regulation, and bladder-filling sensation, among other functions. While research on non-selective cation channels in relation to energy metabolism has primarily focused on TRP channels expressed in primary afferent neurons, studies over the past decade have revealed the important roles of TRP and Piezo channels in brown adipocytes. In this review, we highlight evidence regarding the contributions of TRPV2 and Piezo1 to brown adipocyte differentiation and thermogenesis and briefly summarize recent advances regarding other TRP channels expressed in brown adipocytes. Furthermore, we propose a conceptual framework in which a “modal shift” in TRP/Piezo channels, defined as developmental stage-dependent changes in their functional properties, may contribute to the regulation of brown adipocytes’ functions.

Mechanisms underlying sensory irritation caused by chemical stimuli on the skin surface remain unknown. Recently, it has been clarified that TRPA1 and TRPV1 are important for sensing nociceptive and temperature stimuli. This study aimed to clarify the mechanism of hTRPA1 and hTRPV1 in sensory irritation on the skin by evaluating the effects of anti-bacterial agents, ethanol, or polyols using electro-physiological methods in HEK293 cells. Parabens (1 mM) activated hTRPA1 in an alkyl chain length-dependent manner. Conversely, high concentrations (>500 mM) of ethanol and polyols activate hTRPV1. The intensities of sensory irritation caused by anti-bacterial agents or polyols correlated with their abilities to activate TRPA1 or TRPV1, respectively. Analogs of known TRPA1 antagonists were screened for their TRPA1 inhibitory activity. Arbanol was identified as a novel TRPA1 inhibitor that can be used to reduce sensory irritation on the skin surface. TRP channels are crucial for sensory irritation on the skin surface.

ABSTRACT Transient receptor potential vanilloid 4 (TRPV4) is a Ca 2+ -permeable non-selective cation channel and its activating stimuli include anandamide, bisandrographolide, citric acid, arachidonic acid metabolic products by epoxygenases, hypo-osmotic cell swelling, and warm temperature. TRPV4 is involved in Ca 2+ -dependent signal transduction in several tissues. Since the activation of TRPV4 facilitates adherens junction formation in the skin epithelium, compounds that activate TRPV4 are expected to maintain or improve the barrier function of epidermal cells. In this study, we found that the extract of Arachis hypogaea ( A. hypogaea ) activate human TRPV4 (hTRPV4). In the Ca 2+ -imaging experiment, the application of A. hypogaea extract exhibited an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) in HEK293T cells expressing hTRPV4. The [Ca 2+ ] i increases by application of A. hypogaea extract were not observed in HEK293T cells expressing hTRPV1, mouse TRPV2, hTRPV3, hTRPM8, or hTRPA1. We then examined the physicochemical properties of the components responsible for TRPV4 activation. Ethanol extracts of A. hypogaea caused an increase in [Ca 2+ ] i in hTRPV4-expressing HEK293JN cells, whereas water, chloroform, and hexane extracts showed no activity. Moreover, the application of A. hypogaea extract enhanced transepithelial electrical resistance in the keratinocyte monolayer. These results suggest that A. hypogaea extract may contribute to the maintenance and improvement of the epidermal barrier function.

Background/Objectives: Metabolites produced by gut microbiota play an important role in the crosstalk between the gut and other organs. Although HYA (10-hydroxy-cis-12-octadecenoic acid), a linoleic acid metabolite produced by lactic acid bacteria represented by Lactobacillus, has been shown to exert physiological effects such as metabolic improvement and anti-inflammation in the host, its direct action on adipose tissue and the mechanism remains unknown. Methods: The effect of HYA administration on adipocyte size in mice fed a high-fat diet was examined. In 3T3-L1 mature adipocytes treated with HYA, the amount of intracellular lipid droplets was evaluated by Oil red O staining, gene expression by real-time qPCR, phosphorylation of AMP-activated protein kinase (AMPK) by immunoblotting, and intracellular Ca2+ concentration with calcium imaging. Results: Administration of HYA, but not linoleic acid, to obese mice fed a high-fat diet significantly reduced adipocyte size. To investigate whether the inhibition of adipocyte hypertrophy by HYA has a direct effect on adipocytes, 3T3-L1 adipocytes were treated with HYA, which significantly decreased the amount of intracellular lipid droplets in these cells. Gene expression analysis by real-time PCR showed decreased expression of genes related to lipogenesis such as FAS and ACC1, and increased expression of CPT1A, which is involved in fatty acid oxidation. Mechanistically, HYA was found to activate AMPK in adipocytes by increasing intracellular Ca2+ concentration. Conclusions: HYA suppresses adipocyte hypertrophy by activating AMPK in adipocytes. HYA may be a potential therapeutic for obesity and related metabolic disorders.

Transient receptor potential vanilloid 2 (TRPV2) is a non-selective cation channel activated by mechanical stimuli and temperatures above 52°C. Although we have previously reported that TRPV2 regulates non-shivering thermogenesis through facilitating the expression of genes related to thermogenesis, how TRPV2 activity is regulated in brown adipocytes under physiological conditions remains unclear. Recently, methionine oxidation was reported to lower the activation temperature threshold of TRPV2 to around or even below core body temperature. In the present study, we investigated whether methionine oxidation activates TRPV2 and regulates thermogenesis in the differentiated brown adipocytes. As a result, treatment with Chloramine-T (ChT, a methionine oxidant) activated TRPV2 at temperatures of >30°C in mouse TRPV2-expressing HEK293T cells and in the differentiated brown adipocytes. Moreover, ChT treatment enhanced the expression of genes related to thermogenesis in the differentiated brown adipocytes. These results suggest that methionine oxidation might activate TRPV2 at around body temperature and increase thermogenesis-related gene expression.

Temperature detection and temperature preference are critical for the maintenance of thermal homeostasis and protection from noxious temperature. Methods to measure the time spent on plates of different temperatures such as the 2-plate test and thermal-gradient test are generally used to evaluate temperature preference. In this study, we established a temperature preference test that assesses water drinking behavior by modifying the 2-bottle taste preference test. We found that mice in a common-temperature environment of 23 °C avoid drinking water of temperature more than 40 °C. While mice in a common-temperature environment preferred 10 °C and 30 °C water equally, mice in a hot environment at 35 °C preferred 10 °C water to 30 °C water. Moreover, mice in a cold environment of 10 °C preferred to drink 40 °C water compared to 10 °C water. From these results, the temperature preference test that we developed could be used to evaluate temperature preferences owing to the ambient temperature changes.

Temperature detection is essential for the survival and perpetuation of any species. Thermoreceptors in the skin sense the body temperature and also the temperatures of the ambient air and the objects. In 1997, Dr. David Julius and his colleagues found that a receptor expressed in small-diameter primary sensory neurons was activated by capsaicin (the pungent chemical in hot pepper). This receptor was also activated by temperature above 42 degrees C. That was the first time that a thermal receptor in primary sensory neurons has been identified. This receptor is named transient receptor potential vanilloid 1 (TRPV1). Now, 11 thermosensitive TRP channels are known. In this chapter, we summarize the reports and analyze thermosensitive TRP channels in a variety of ways to clarify the activation mechanisms by which temperature changes are sensed.

Temperature detection is essential for the survival and perpetuation of any species. Thermoreceptors in the skin sense body temperature as well as the temperatures of ambient air and objects. Since Dr. David Julius and his colleagues discovered that TRPV1 is expressed in small-diameter primary sensory neurons, and activated by temperatures above 42 °C, 11 of thermo-sensitive TRP channels have been identified. TRPM3 expressed in sensory neurons acts as a sensor for noxious heat. TRPM4 and TRPM5 are Ca2⁺-activated monovalent cation channels, and their activity is drastically potentiated by temperature increase. This review aims to summarize the expression patterns, electrophysiological properties, and physiological roles of TRPM3, TRPM4, and TRPM5 associated with thermosensation.

Transient receptor potential (TRP) channels play a significant role in taste perception. TRP ankyrin 1 (TRPA1) is present in the afferent sensory neurons and is activated by food-derived ingredients, such as Japanese horseradish, cinnamon, and garlic. The present study aimed to investigate the expression of TRPA1 in taste buds, and determine its functional roles in taste perception using TRPA1-deficient mice. In circum-vallate papillae, TRPA1 immunoreactivity colocalised with P2X2 receptor-positive taste nerves but not with type II or III taste cell markers. Behavioural studies showed that TRPA1 deficiency significantly reduced sensitivity to sweet and umami tastes, but not to salty, bitter, and sour tastes, compared to that in wild-type animals. Furthermore, administration of the TRPA1 antagonist HC030031 significantly decreased taste preference to sucrose solution compared to that in the vehicle-treated group in the two-bottle preference tests. TRPA1 deficiency did not affect the structure of circumvallate papillae or the expression of type II or III taste cell and taste nerve markers. Adenosine 5'-O-(3-thio)triphosphate evoked inward currents did not differ between P2X2-and P2X2/TRPA1-expressing human embryonic kidney 293T cells. TRPA1-deficient mice had significantly decreased c-fos expression in the nucleus of the solitary tract in the brain stem following sucrose stimulation than wild-type mice. Taken together, the current study suggested that TRPA1 in the taste nerve contributes to the sense of sweet taste in mice.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective cation channel that is activated by a variety of stimuli and acts as a nociceptor. Mouse and human TRPA1 exhibit different reactivity to some stimuli, including chemicals such as menthol as well as cold stimuli. The cold sensitivity of TRPA1 in mammalian species is controversial. Here, we analyzed the reactivity of heterologously expressed canine TRPA1 as well as the mouse and human orthologs to menthol or cold stimulation in Ca2+-imaging experiments. Canine and human TRPA1 exhibited a similar response to menthol, that is, activation in a concentration-dependent manner, even at the high concentration range in contrast to the mouse ortholog, which did not respond to high concentration of menthol. In addition, the response during the removal of menthol was different; mouse TRPA1-expressing cells exhibited a typical response with a rapid and clear increase in [Ca2+](i) ("off-response"), whereas [Ca2+](i) in human TRPA1-expressing cells was dramatically decreased by the washout of menthol and [Ca2+](i) in canine TRPA1-expressing cells was slightly decreased. Finally, canine TRPA1 as well as mouse and human TRPA1 were activated by cold stimulation (below 19-20 degrees C). The sensitivity to cold stimulation differed between these species, that is, human TRPA1 activated at higher temperatures compared with the canine and mouse orthologs. All of the above responses were suppressed by the selective TRPA1 inhibitor HC-030031. Because the concentration-dependency and "off-response" of menthol as well as the cold sensitivity were not uniform among these species, studies of canine TRPA1 might be useful for understanding the species-specific functional properties of mammalian TRPA1.

Low-temperature-induced fatty acid desaturation is highly conserved in animals, plants, and bacteria. Allyl isothiocyanate (AITC) is an agonist of the transient receptor potential ankyrin 1 (TRPA1), which is activated by various chemophysiological stimuli, including low temperature. However, whether AITC induces fatty acid desaturation remains unknown. We showed here that AITC increased levels of glycerophospholipids (GP) esterified with unsaturated fatty acids, especially docosahexaenoic acid (DHA) in TRPA1-expressing HEK cells. Additionally, GP-DHA including phosphatidylcholine (18:0/22:6) and phosphatidylethanolamine (18:0/22:6) was increased in the brain and liver of AITC-administered mice. Moreover, intragastrical injection of AITC in ovariectomized (OVX) female C57BL/6J mice dose-dependently shortened the Δlatency time determined by the Morris water maze test, indicating AITC ameliorated the cognitive function decline in these mice. Thus, the oral administration of AITC maintains GP-DHA in the liver and brain, proving to be a potential strategy for preventing cognitive decline.

Transient receptor potential ankyrin 1 (TRPA1) is a Ca2+-permeable, nonselective cation channel that is activated by a wide variety of stimuli. Mouse and human TRPA1 exhibit different reactivity to some stimuli, including chemicals such as menthol as well as cold stimuli. In this study, we analyzed the reactivity of heterologously expressed canine, mouse, and human TRPA1 to menthol or cold stimulation in Ca2+-imaging experiments. Canine and human TRPA1 exhibited a similar response to menthol, namely, activation in a concentration-dependent manner, even at the high concentration range, in contrast to the mouse TRPA1. In addition, the response during the removal of menthol was different; mouse TRPA1 exhibited a typical response with a rapid increase in [Ca2+]i (“off-response”), whereas canine and human TRPA1 responded differently to each other. Finally, canine TRPA1 as well as mouse and human TRPA1 were activated by cold stimulation, although cold sensitivity varied among these species. These responses were suppressed by the selective TRPA1 inhibitor HC-030031. Because the concentration-dependency and “off-response” of menthol as well as the cold sensitivity were not uniform among these species, studies of canine TRPA1 might be useful for understanding the species-specific functional properties of mammalian TRPA1.

Chronic constipation is prevalent and involves both colon sensitivity and various changes in intestinal bacteria, particularly mucosa-associated microflora. Here we examined regulatory mechanisms of TRPV4 expression by co-culturing colon epithelial cell lines with intestinal bacteria and their derivatives. We also investigated TRPV4 expression in colon epithelium from patients with constipation. Colon epithelial cell lines were co-cultured with various enterobacteria (bacterial components and supernatant), folate, LPS, or short chain fatty acids. TRPV4 expression levels and promoter DNA methylation were assessed using pyrosequencing, and microarray network analysis. For human samples, correlation coefficients were calculated and multiple regression analyses were used to examine the association between clinical background, rectal TRPV4 expression level and mucosa-associated microbiota. Co-culture of CCD841 cells with P. acnes, C. perfringens, or S. aureus transiently decreased TRPV4 expression but did not induce methylation. Co-culture with clinical isolates and standard strains of K. oxytoca, E. faecalis, or E. coli increased TRPV4 expression in CCD841 cells, and TRPV4 and TNF-alpha expression were increased by E. coli culture supernatants but not bacterial components. Although folate, LPS, IL-6, TNF-alpha, or SCFAs alone did not alter TRPV4 expression, TRPV4 expression following exposure to E. coli culture supernatants was inhibited by butyrate or TNF-alphaR1 inhibitor and increased by p38 inhibitor. Microarray network analysis showed activation of TNF-alpha, cytokines, and NOD signaling. TRPV4 expression was higher in constipated patients from the terminal ileum to the colorectum, and multiple regression analyses showed that low stool frequency, frequency of defecation aids, and duration were associated with TRPV4 expression. Meanwhile, incomplete defecation, time required to defecate, and number of defecation failures per 24 h were associated with increased E. faecalis frequency. Colon epithelium cells had increased TRPV4 expression upon co-culture with K. oxytoca, E. faecalis, or E. coli supernatants, as well as TNFα-stimulated TNFαR1 expression via a pathway other than p38. Butyrate treatment suppressed this increase. Epithelial TRPV4 expression was increased in constipated patients, suggesting that TRPV4 together with increased frequency of E. faecalis may be involved in the pathogenesis of various constipation symptoms.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable non-selective cation channel and its activating stimuli include anandamide, bisandrographolide, citric acid, arachidonic acid metabolic products by epoxygenases, hypo-osmotic cell swelling, and warm temperature. TRPV4 is involved in Ca2+-dependent signal transduction in several tissues. Since the activation of TRPV4 facilitates adherence-junction formation in the skin epithelium, compounds that activate TRPV4 are expected to maintain or improve the barrier function of epidermal cells. In this study, we found that the extract of Arachis hypogaea (A. hypogaea) activates human TRPV4 (hTRPV4). In the Ca2+-imaging experiment, the application of A. hypogaea extract exhibited the intracellular Ca2+ concentration ([Ca2+]i) increases in HEK293T cells expressing hTRPV4. The [Ca2+]i increases by application of A. hypogaea extract were not observed in HEK293T cells expressing TRPV1, TRPV2, TRPV3, TRPM8, or TRPA1. Moreover, the application of A. hypogaea extract enhanced transepithelial electrical resistance in the keratinocyte monolayer. These results suggest that A. hypogaea extract is useful for maintaining and improving the barrier function of epidermal cells.

Brown adipocytes cause an energy consumption by heat production and are thought to be a target for the prevention of obesity and related metabolic disorders. Piezo1 is a Ca2+-permeable non-selective cation channel and activated by mechanical stimuli. While, Piezo1 has been reported to be involved in mechano-sensation in non-sensory tissues, the expression and role of Piezo1 in brown adipocytes have not been well clarified. In this study, we evaluated a brown adipocytes line from UCP1-mRFP1 transgenic mice and analyzed this cell. Application of Yoda-1, a Piezo1 agonistsuppressed brown adipocytes differentiation in a dose-dependent manner. This suppression was significantly recovered by co-application with a Piezo1 antagonist and a calcineurin inhibitor. In addition, knock-down of Piezo1 impaired Yoda-1-induced suppression of brown adipocyte differentiation and application of Yoda-1 enhanced the calcineurin activity. These results suggest that activation of Piezo1 might suppress the differentiation through calcineurin pathway in brown pre-adipocytes.

Although menthol generally provides a pleasant cooling sensation in humans, at high concentrations it can cause discomfort and/or pain. This effect is attributable to two cation channels, TRPM8 and TRPA1, that exhibit different dose dependency in humans. Given that the dose dependency of these receptors to menthol might differ in other mammalian species, we examined the reactivity of canine TRPM8 and TRPA1 to menthol by using recombinantly expressed channels. HEK293T cells were transfected with canine TRPM8 or TRPA1 and subjected to calcium imaging. Canine TRPA1 was confirmed to be activated by the selective TRPA1 agonist allyl isothiocyanate in a dose-dependent manner, which was inhibited by the TRPA1 antagonist HC-030031. Canine TRPA1 was also activated by menthol in a dose-dependent manner, even at high concentrations (up to 3 mM), in contrast with TRPA1 in rodents, which was inhibited by a high concentration of menthol. Canine TRPA1 showed a much higher value at EC50 for menthol response compared with canine TRPM8 (178.9 μM and 5.3 μM, respectively). The activation of TRPA1 by menthol was inhibited by HC-030031 but it was not inhibited by the TRPM8 antagonist RQ-00203078. Our results suggest that menthol activates TRPM8 at low concentrations and may induce a pleasant cooling sensation, whereas at high concentrations it activates TRPA1 and may cause discomfort in dogs as in humans. In addition, this mechanism is suggested to be conserved in some carnivora and primates.

Brown adipocytes expend energy via heat production and are a potential target for the prevention of obesity and related metabolic disorders. Piezo1 is a Ca 2+ -permeable non-selective cation channel activated by mechanical stimuli. Piezo1 is reported to be involved in mechano-sensation in non-sensory tissues. However, the expression and roles of Piezo1 in brown adipocytes have not been well clarified. Here, we generated a brown adipocyte line derived from UCP1-mRFP1 transgenic mice and showed that Piezo1 is expressed in pre-adipocytes. Application of Yoda-1, a Piezo1 agonist, suppressed brown adipocyte differentiation, and this suppression was significantly attenuated by treatment with a Piezo1 antagonist and by Piezo1 knockdown. Furthermore, the suppression of brown adipocyte differentiation by Yoda-1 was abolished by co-treatment with a calcineurin inhibitor. Thus, these results suggest that activation of Piezo1 suppresses brown adipocyte differentiation via the calcineurin pathway.

TRPA1 is a non-selective cation channel and has been shown to be activated by a wide variety of noxious compounds and physiological stressors. Interestingly, TRPA1 is reported to be stimulated by menthol, an agonist of TRPM8, with different dose dependencies between mouse and human. It has been suggested that this different reactivity may be attributable to three amino acid residues in the TM5 region of TRPA1, namely, S–T–V in human and S–T–G in mouse. In this study, to further investigate the importance of the TM5 region through comparison with other mammalian species, canine TRPA1 cDNA was cloned and its reactivity to several agonists was compared in dog and mouse by using recombinant proteins. The TM5 region of cloned canine TRPA1 showed high similarity to that of human TRPA1 and the S–T–V residues were conserved. HEK293T cells were transfected with mouse or canine TRPA1 and subjected to calcium influx imaging. Both mouse and canine TRPA1 were activated by the TRPA1 agonist allyl isothiocyanate and were inactivated by the TRPA1 antagonist HC-030031. In contrast, reactivity to menthol was observed to differ between these two species. Mouse TRPA1 was activated by 100 µM of menthol and showed transient Ca2+ influx when menthol was washed out ("off response"), whereas canine TRPA1 activation required a high (300 µM) concentration of menthol, but no off response was observed. These results showed that the reactivity of canine TRPA1 against menthol is similar to that of human TRPA1 but not to mouse TRPA1, which might reflect the similarity of their respective TM5 regions.

The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent-permeable cation channel that is activated by intracellular Ca2+. Expression of TRPM5 has been shown in taste cells, pancreas, brainstem and olfactory epithelium, and this channel is thought to be involved in controlling membrane potentials. In whole-cell patch-clamp recordings, TRPM5 exhibited voltage-dependent inactivation at negative membrane potentials and time constant of voltage-dependent inactivation of TRPM5 did not depend on the intracellular Ca2+ concentrations between 100 and 500 nM. Alanine substitution at Y913 and I916 in the pore helix of TRPM5 increased time constant of voltage-dependent inactivation. Meanwhile, voltage-dependent inactivation was reduced in TRPM5 mutants having glycine substitution at L901, Y913, Q915 and I916 in the pore helix. From these results, we conclude that the pore helix in the outer pore loop might play a role in voltage-dependent inactivation of TRPM5.