Phosphoinositide Regulation of TRP Channels: A Functional Overview in the Structural Era.

Transient receptor potential (TRP) ion channels have diverse activation mechanisms including physical stimuli, such as high or low temperatures, and a variety of intracellular signaling molecules. Regulation by phosphoinositides and their derivatives is their only known common regulatory feature. For most TRP channels, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] serves as a cofactor required for activity. Such dependence on PI(4,5)P2 has been demonstrated for members of the TRPM subfamily and for the epithelial TRPV5 and TRPV6 channels. Intracellular TRPML channels show specific activation by PI(3,5)P2. Structural studies uncovered the PI(4,5)P2 and PI(3,5)P2 binding sites for these channels and shed light on the mechanism of channel opening. PI(4,5)P2 regulation of TRPV1-4 as well as some TRPC channels is more complex, involving both positive and negative effects. This review discusses the functional roles of phosphoinositides in TRP channel regulation and molecular insights gained from recent cryo-electron microscopy structures.

Separation of Oral Cooling and Warming Requires TRPM8.

Cooling sensations arise inside the mouth during ingestive and homeostasis behaviors. Oral presence of cooling temperature engages the cold and menthol receptor TRPM8 (transient receptor potential melastatin 8) on trigeminal afferents. Yet, how TRPM8 influences brain and behavioral responses to oral temperature is undefined. Here we used in vivo neurophysiology to record action potentials stimulated by cooling and warming of oral tissues from trigeminal nucleus caudalis neurons in female and male wild-type and TRPM8 gene deficient mice. Using these lines, we also measured orobehavioral licking responses to cool and warm water in a novel, temperature-controlled fluid choice test. Capture of antidromic electrophysiological responses to thalamic stimulation identified that wild-type central trigeminal neurons showed diverse responses to oral cooling. Some neurons displayed relatively strong excitation to cold <10°C (COLD neurons) while others responded to only a segment of mild cool temperatures below 30°C (COOL neurons). Notably, TRPM8 deficient mice retained COLD-type but lacked COOL cells. This deficit impaired population responses to mild cooling temperatures below 30°C and allowed warmth-like (≥35°C) neural activity to pervade the normally innocuous cool temperature range, predicting TRPM8 deficient mice would show anomalously similar orobehavioral responses to warm and cool temperatures. Accordingly, TRPM8 deficient mice avoided both warm (35°C) and mild cool (≤30°C) water and sought colder temperatures in fluid licking tests, whereas control mice avoided warm but were indifferent to mild cool and colder water. Results imply TRPM8 input separates cool from warm temperature sensing and suggest other thermoreceptors also participate in oral cooling sensation.

The acquisition of cold sensitivity during TRPM8 ion channel evolution.

To cope with temperature fluctuations, molecular thermosensors in animals play a pivotal role in accurately sensing ambient temperature. Transient receptor potential melastatin 8 (TRPM8) is the most established cold sensor. In order to understand how the evolutionary forces bestowed TRPM8 with cold sensitivity, insights into both emergence of cold sensing during evolution and the thermodynamic basis of cold activation are needed. Here, we show that the trpm8 gene evolved by forming and regulating two domains (MHR1-3 and pore domains), thus determining distinct cold-sensitive properties among vertebrate TRPM8 orthologs. The young trpm8 gene without function can be observed in the closest living relatives of tetrapods (lobe-finned fishes), while the mature MHR1-3 domain with independent cold sensitivity has formed in TRPM8s of amphibians and reptiles to enable channel activation by cold. Furthermore, positive selection in the TRPM8 pore domain that tuned the efficacy of cold activation appeared late among more advanced terrestrial tetrapods. Interestingly, the mature MHR1-3 domain is necessary for the regulatory mechanism of the pore domain in TRPM8 cold activation. Our results reveal the domain-based evolution for TRPM8 functions and suggest that the acquisition of cold sensitivity in TRPM8 facilitated terrestrial adaptation during the water-to-land transition.

Endogenous Inflammatory Mediators Produced by Injury Activate TRPV1 and TRPA1 Nociceptors to Induce Sexually Dimorphic Cold Pain That Is Dependent on TRPM8 and GFRα3.

The detection of environmental temperatures is critical for survival, yet inappropriate responses to thermal stimuli can have a negative impact on overall health. The physiological effect of cold is distinct among somatosensory modalities in that it is soothing and analgesic, but also agonizing in the context of tissue damage. Inflammatory mediators produced during injury activate nociceptors to release neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P, inducing neurogenic inflammation, which further exasperates pain. Many inflammatory mediators induce sensitization to heat and mechanical stimuli but, conversely, inhibit cold responsiveness, and the identity of molecules inducing cold pain peripherally is enigmatic, as are the cellular and molecular mechanisms altering cold sensitivity. Here, we asked whether inflammatory mediators that induce neurogenic inflammation via the nociceptive ion channels TRPV1 (vanilloid subfamily of transient receptor potential channel) and TRPA1 (transient receptor potential ankyrin 1) lead to cold pain in mice. Specifically, we tested cold sensitivity in mice after intraplantar injection of lysophosphatidic acid or 4-hydroxy-2-nonenal, finding that each induces cold pain that is dependent on the cold-gated channel transient receptor potential melastatin 8 (TRPM8). Inhibition of CGRP, substance P, or toll-like receptor 4 (TLR4) signaling attenuates this phenotype, and each neuropeptide produces TRPM8-dependent cold pain directly. Further, the inhibition of CGRP or TLR4 signaling alleviates cold allodynia differentially by sex. Last, cold pain induced by both inflammatory mediators and neuropeptides requires TRPM8, as well as the neurotrophin artemin and its receptor GDNF receptor α3 (GFRα3). These results are consistent with artemin-induced cold allodynia requiring TRPM8, demonstrating that neurogenic inflammation alters cold sensitivity via localized artemin release that induces cold pain via GFRα3 and TRPM8.SIGNIFICANCE STATEMENT The cellular and molecular mechanisms that generate pain are complex with a diverse array of pain-producing molecules generated during injury that act to sensitize peripheral sensory neurons, thereby inducing pain. Here we identify a specific neuroinflammatory pathway involving the ion channel TRPM8 (transient receptor potential cation channel subfamily M member 8) and the neurotrophin receptor GFRα3 (GDNF receptor α3) that leads to cold pain, providing select targets for potential therapies for this pain modality.

The powerful potential of amino acid menthyl esters for anti-inflammatory and anti-obesity therapies.

Our newly developed menthyl esters of valine and isoleucine exhibit anti-inflammatory properties beyond those of the well-known menthol in macrophages stimulated by lipopolysaccharide (LPS) and in a mouse model of colitis induced by sodium dextran sulfate. Unlike menthol, which acts primarily through the cold-sensitive TRPM8 channel, these menthyl esters displayed unique mechanisms that operate independently of this receptor. They readily penetrated target cells and efficiently suppressed LPS-stimulated tumour necrosis factor-alpha (Tnf) expression mediated by liver X receptor (LXR), a key nuclear receptor that regulates intracellular cholesterol and lipid balance. The menthyl esters showed affinity for LXR and enhanced the transcriptional activity through their non-competitive and potentially synergistic agonistic effect. This effect can be attributed to the crucial involvement of SCD1, an enzyme regulated by LXR, which is central to lipid metabolism and plays a key role in the anti-inflammatory response. In addition, we discovered that the menthyl esters showed remarkable efficacy in suppressing adipogenesis in 3T3-L1 adipocytes at the mitotic clonal expansion stage in an LXR-independent manner as well as in mice subjected to diet-induced obesity. These multiple capabilities of our compounds establish them as formidable allies in the fight against inflammation and obesity, paving the way for a range of potential therapeutic applications.

Residues of TRPM8 at the Lipid-Water-Interface have Coevolved with Cholesterol Interaction and are Relevant for Diverse Health Disorders.

TRPM8 is a non-selective cation channel that is expressed in several tissues and cells and also has a unique property to be activated by low-temperature. In this work, we have analyzed the conservation of amino acids that are present in the lipid-water-interface (LWI) region of TRPM8, the region which experiences a microenvironment near the membrane surface. We demonstrate that the amino acids present in the LWI region are more conserved than the transmembrane or even full-length TRPM8, suggesting strong selection pressure in these residues. TRPM8 also has several conserved cholesterol-binding motifs where cholesterol can bind in different modes and energies. We suggest that mutations and/or physiological conditions can potentially alter these TRPM8-cholesterol complexes and can lead to physiological disorders or even apparently irreversible diseases such as cancer and neurodegeneration.

Topical menthol, a pharmacological cold mimic, induces cold sensitivity, adaptive thermogenesis and brown adipose tissue activation in mice.

AIM: Brown adipose tissue (BAT) thermogenesis has profound energy-expanding potential, which makes it an attractive target tissue to combat ever-increasing obesity and its other associated metabolic complications. Although it is fairly accepted that cold is a potent inducer of BAT activation and function, there are limited studies on the mechanisms of pharmacological cold-mimicking agents, such as the TRPM8 agonist, menthol, on BAT thermogenesis and activation. METHODS: Herein, we sought to determine the effect of topical application of menthol (10% w/v [4 g/kg] cream formulation/day for 15 days) on temperature sensitivity behaviour (thermal gradient assay, nesting behaviour), adaptive thermogenesis (infrared thermography, core body temperature), BAT sympathetic innervation (tyrosine hydroxylase immunohistochemistry) and activation (18F-FDG PET-CT analysis, Uncoupling Protein 1 immunohistochemistry and BAT gene expression), whole-body energy expenditure (indirect calorimetry) and other metabolic variables in male C57BL/6N mice. RESULTS: We show that male C57BL/6N mice: (a) develop a warm-seeking and cold-avoiding thermal preference phenotype; (b) display increased locomotor activity and adaptive thermogenesis; (c) show augmented sympathetic innervation in BAT and its activation; (d) exhibit enhanced gluconeogenic capacity (increased glucose excursion in response to pyruvate) and insulin sensitivity; and (e) show enhanced whole-body energy expenditure and induced lipid-utilizing phenotype after topical menthol application. CONCLUSIONS: Taken together, our findings highlight that pharmacological cold mimicking using topical menthol application presents a potential therapeutic strategy to counter weight gain and related complications.

Menthol alleviates post-race elevations in muscle soreness and metabolic and respiratory stress during running.

PURPOSE: We evaluated (1) whether participating in middle- and long-distance running races augments muscle soreness, oxygen cost, respiration, and exercise exertion during subsequent running, and (2) if post-race menthol application alleviates these responses in long-distance runners. METHODS: Eleven long-distance runners completed a 1500-m race on day 1 and a 3000-m race on day 2. On day 3 (post-race day), either a 4% menthol solution (Post-race menthol) or a placebo solution (Post-race placebo) serving as a vehicle control, was applied to their lower leg skin, and their perceptual and physiological responses were evaluated. The identical assessment with the placebo solution was also conducted without race participation (No-race placebo). RESULTS: The integrated muscle soreness index increased in the Post-race placebo compared to the No-race placebo (P < 0.001), but this response was absent in the Post-race menthol (P = 0.058). Oxygen uptake during treadmill running tended to be higher (4.3%) in the Post-race placebo vs. No-race placebo (P = 0.074). Oxygen uptake was 5.4% lower in the Post-race menthol compared to the Post-race placebo (P = 0.018). Minute ventilation during treadmill running was 6.7-7.6% higher in the Post-race placebo compared to No-race placebo, whereas it was 6.6-9.0% lower in the Post-race menthol vs. Post-race placebo (all P ≤ 0.001). The rate of perceived exertion was 7.0% lower in the Post-race menthol vs. Post-race placebo (P = 0.007). CONCLUSIONS: Middle- and long-distance races can subsequently elevate muscle soreness and induce respiratory and metabolic stress, but post-race menthol application to the lower legs can mitigate these responses and reduce exercise exertion in long-distance runners.

Thermosensation and TRP Channels.

Somatosensory neurons can sense external temperature by converting sensation of temperature information to neural activity via afferent input to the central nervous system. Various populations of somatosensory neurons have specialized gene expression, including expression of thermosensitive transient receptor potential (TRP) ion channels. Thermosensitive TRP channels are responsible for thermal transduction at the peripheral ends of somatosensory neurons and can sense a wide range of temperatures. Here we focus on several thermosensitive TRP channels including TRPV1, TRPV4, TRPM2, TRPM3, TRPM8, TRPC5, and TRPA1 in sensory neurons. TRPV3, TRPV4, and TRPC5 are also involved in somatosensation in nonneuronal cells and tissues. In particular, we discuss whether skin senses ambient temperatures through TRPV3 and TRPV4 activation in skin keratinocytes and the involvement of TRPM2 expressed by hypothalamic neurons in thermosensation in the brain.

Roles of Thermosensitive Transient Receptor Channels TRPV1 and TRPM8 in Paclitaxel-Induced Peripheral Neuropathic Pain.

Paclitaxel, a microtubule-stabilizing chemotherapy drug, can cause severe paclitaxel-induced peripheral neuropathic pain (PIPNP). The roles of transient receptor potential (TRP) ion channel vanilloid 1 (TRPV1, a nociceptor and heat sensor) and melastatin 8 (TRPM8, a cold sensor) in PIPNP remain controversial. In this study, Western blotting, immunofluorescence staining, and calcium imaging revealed that the expression and functional activity of TRPV1 were upregulated in rat dorsal root ganglion (DRG) neurons in PIPNP. Behavioral assessments using the von Frey and brush tests demonstrated that mechanical hyperalgesia in PIPNP was significantly inhibited by intraperitoneal or intrathecal administration of the TRPV1 antagonist capsazepine, indicating that TRPV1 played a key role in PIPNP. Conversely, the expression of TRPM8 protein decreased and its channel activity was reduced in DRG neurons. Furthermore, activation of TRPM8 via topical application of menthol or intrathecal injection of WS-12 attenuated the mechanical pain. Mechanistically, the TRPV1 activity triggered by capsaicin (a TRPV1 agonist) was reduced after menthol application in cultured DRG neurons, especially in the paclitaxel-treated group. These findings showed that upregulation of TRPV1 and inhibition of TRPM8 are involved in the generation of PIPNP, and they suggested that inhibition of TRPV1 function in DRG neurons via activation of TRPM8 might underlie the analgesic effects of menthol.

Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun.

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the ßγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.

Genomic adaptation to extreme climate conditions in beef cattle as a consequence of cross-breeding program.

BACKGROUND: Understanding the evolutionary forces related to climate changes that have been shaped genetic variation within species has long been a fundamental pursuit in biology. In this study, we generated whole-genome sequence (WGS) data from 65 cross-bred and 45 Mongolian cattle. Together with 62 whole-genome sequences from world-wide cattle populations, we estimated the genetic diversity and population genetic structure of cattle populations. In addition, we performed comparative population genomics analyses to explore the genetic basis underlying variation in the adaptation to cold climate and immune response in cross-bred cattle located in the cold region of China. To elucidate genomic signatures that underlie adaptation to cold climate, we performed three statistical measurements, fixation index (FST), log2 nucleotide diversity (θπ ratio) and cross population composite likelihood ratio (XP-CLR), and further investigated the results to identify genomic regions under selection for cold adaptation and immune response-related traits. RESULTS: By generating WGS data, we investigated the population genetic structure and phylogenetic relationship of studied cattle populations. The results revealed clustering of cattle groups in agreement with their geographic distribution. We detected noticeable genetic diversity between indigenous cattle ecotypes and commercial populations. Analysis of population structure demonstrated evidence of shared genetic ancestry between studied cross-bred population and both Red-Angus and Mongolian breeds. Among all studied cattle populations, the highest and lowest levels of linkage disequilibrium (LD) per Kb were detected in Holstein and Rashoki populations (ranged from ~ 0.54 to 0.73, respectively). Our search for potential genomic regions under selection in cross-bred cattle revealed several candidate genes related with immune response and cold shock protein on multiple chromosomes. We identified some adaptive introgression genes with greater than expected contributions from Mongolian ancestry into Molgolian x Red Angus composites such as TRPM8, NMUR1, PRKAA2, SMTNL2 and OXR1 that are involved in energy metabolism and metabolic homeostasis. In addition, we detected some candidate genes probably associated with immune response-related traits. CONCLUSION: The study identified candidate genes involved in responses to cold adaptation and immune response in cross-bred cattle, including new genes or gene pathways putatively involved in these adaptations. The identification of these genes may clarify the molecular basis underlying adaptation to extreme environmental climate and as such they might be used in cattle breeding programs to select more efficient breeds for cold climate regions.

Modulation of TRPM8 alters the phagocytic activity of microglia and induces changes in sub-cellular organelle functions.

In this work, we investigated the presence and function of TRPM8, a non-selective and cold-sensitive Ca2+-permeable ion channel in the primary microglia cell as well as in microglia cell line BV2. We demonstrate that primary microglia as well as BV2 express TRPM8 endogenously. Both pharmacological activation or inhibition of TRPM8 causes enhanced uptake of bacterial particles at early time points of infection. In BV2, TRPM8 activation and/or LPS-signaling alters its surface expression and cytosolic ROS production. TRPM8 modulation in the absence and presence of LPS causes differential regulation of cytosolic pH and lysosomal pH. Notably, TRPM8 modulation also alters the correlation between lysosomal pH and cytosolic pH depending on TRPM8 modulation and the presence or absence of LPS. Collectively our data suggest that TRPM8 is involved in the regulation of subcellular organelle, i.e. mitochondrial and lysosomal functions. Data also suggest that primarily TRPM8 activation, but often deviation from endogenous TRPM8 function is linked with better innate immune function mediated by microglial cells. We suggest that TRPM8-mediated regulations of sub-cellular organelle functions are more context-dependent manner. Such understanding is relevant in the context of microglial cell functions and innate immunity.

Topical application of the pharmacological cold mimetic menthol stimulates brown adipose tissue thermogenesis through a TRPM8, UCP1, and norepinephrine dependent mechanism in mice housed at thermoneutrality.

Increasing whole-body energy expenditure via the pharmacological activation of uncoupling protein 1 (UCP1)-dependent brown adipose tissue (BAT) thermogenesis is a promising weight management strategy, yet most therapeutics studied in rodents to date either induce compensatory increases in energy intake, have thermogenic effects that are confounded by sub-thermoneutral housing temperatures or are not well tolerated in humans. Here, we sought to determine whether the non-invasive topical application of the pharmacological cold mimetic and transient receptor potential (TRP) cation channel subfamily M member 8 (TRPM8) agonist L-menthol (MNTH), could be used to stimulate BAT thermogenesis and attenuate weight gain in mice housed at thermoneutrality. Using three different strains of mice and multiple complimentary approaches to quantify thermogenesis in vivo, coupled with ex vivo models to quantify direct thermogenic effects, we were able to convincingly demonstrate the following: (1) acute topical MNTH application induces BAT thermogenesis in a TRPM8- and UCP1-dependent manner; (2) MNTH-induced BAT thermogenesis is sufficient to attenuate weight gain over time without affecting energy intake in lean and obese mice; (3) the ability of topical MNTH application to stimulate BAT thermogenesis is mediated, in part, by a central mechanism involving the release of norepinephrine. These data collectively suggest that topical application of MNTH may be a promising weight management strategy.

Downregulation of TRPM7, TRPM8, and TRPV1 channels modulate apoptotic parameters and neurodegenerative markers: Focus on neuronal differentiation and Parkinson's disease model.

The transient receptor potential channel (TRP) channels are expressed in neuronal tissues and involved in neurological diseases such as pain, epilepsy, neuronal apoptosis, and neurodegenerative diseases. Formerly, we have investigated how neuronal differentiation changes TRP channels expression profile and how Parkinson's disease model is related with this expression levels. We have found that transient receptor potential channel melastatin subtype 7 (TRPM7), transient receptor potential channel melastatin subtype 8 and transient receptor potential channel vanilloid subtype 1 (TRPV1) channels have pivotal effects on differentiation and 1-Methyl-4-phenylpyridinium (MPP+ )-induced Parkinson's disease model in SH-SY5Y cells. In this study, we have investigated that downregulation of the TRP channels to evaluate how differentiation status changes to Parkinson's disease pathological hallmarks. We have also performed to other analyses to elucidate these TRP channels' function in MPP+ -induced neurotoxicity related apoptosis, cell viability, caspase 3 and 9 enzyme activities, intracellular reactive oxygen species production, mitochondrial depolarization levels, Ca2+ signaling, Alpha-synuclein and Dopamine levels, mono amino oxidase A and B enzymatic activities, both in differentiated and undifferentiated neuronal cells. Herein we have concluded that especially TRPM7 and TRPV1 channels have distinct role in Parkinson's disease pathology via their activity changings in pathological state, and downregulation of these channels or specific antagonists can be useful for the possible treatment strategy for Parkinson's disease and related markers.

Efficacy of KPR-5714, a selective transient receptor potential melastatin 8 antagonist, on chronic psychological stress-induced bladder overactivity in male rats.

AIMS: Chronic psychological stress aggravates lower urinary tract symptoms. Among others, water avoidance stress is a chronic psychological stressor that plays a causal role in the exacerbation and development of bladder dysfunction in rats. In this report, the effects of KPR-5714, which is a selective transient receptor potential melastatin 8 (TRPM8) antagonist, on bladder overactivity induced by water avoidance stress were examined. METHODS: Male rats were subjected to water avoidance stress for 2 h per day for 10 consecutive days. The effects of water avoidance stress on voiding behavior using metabolic cages and histological bladder changes were investigated in rats. The involvement of bladder C-fiber afferent on voiding frequency in rats exposed to water avoidance stress was assessed using capsaicin. The effects of KPR-5714 on storage dysfunction in rats subjected to water avoidance stress were examined. RESULTS: In voiding behavior measurements, water avoidance stress-induced storage dysfunction, causing a decrease in the mean voided volume and increasing voiding frequency. A comparison of bladders from normal rats and rats exposed to water avoidance stress showed no histological differences. Water avoidance stress-induced bladder overactivity was completely inhibited by pretreatment with capsaicin. KPR-5714 showed a tendency to increase the mean voided volume and significantly decreased the voiding frequency without affecting the total voided volume in these rats. CONCLUSION: The results suggest that KPR-5714 is a promising option for treating chronic psychological stress-induced bladder overactivity.

TRPA1/M8 agonists upregulate ciliary beating through the pannexin-1 channel in the human nasal mucosa.

BACKGROUND: Nasal breathing is important for maintaining physiological respiration. However, airflow in the nasal cavity has an inherent cooling effect and may suppress ciliary beating, an essential frontline defense in the airway. Nasal airflow is thought to be perceived by thermoreceptors for cool temperatures. We herein investigated the effect of the activation of thermosensitive transient receptor potentials (TRPs) for cool/cold temperatures on ciliary beating to search for a compensatory mechanism. METHODS: Inferior turbinates were collected from patients with chronic hypertrophic rhinitis. Ex vivo ciliary beat frequency (CBF) and ATP release were measured using a high-speed digital video camera and by luciferin-luciferase assay, respectively. Intracellular Ca2+ ([Ca2+]i) imaging of isolated ciliated cells was performed using Fluo-8. The nasal mucosae were also subjected to fluorescence immunohistochemistry and real-time RT-PCR for TRPA1/TRPM8. RESULTS: CBF was significantly increased by adding either cinnamaldehyde (TRPA1 agonist) or l-menthol (TRPM8 agonist). This increase was inhibited by pannexin-1 blockers, carbenoxolone and probenecid. Cinnamaldehyde and l-menthol also increased the ATP release from the nasal mucosa and [Ca2+]i of isolated ciliated cells. Immunohistochemistry detected TRPA1 and TRPM8 on the epithelial surface including the cilia and in the submucosal nasal glands. Existence of these receptors were confirmed at the transcriptional level by real-time RT-PCR. CONCLUSIONS: These results indicate the stimulatory effect of the activation of TRPA1/TRPM8 on ciliary beating in the nasal mucosa, which would be advantageous to maintain airway mucosal defense against the fall of temperature under normal nasal breathing. This stimulatory effect is likely to be mediated by pannexin-1.

Why wild giant pandas frequently roll in horse manure.

Attraction to feces in wild mammalian species is extremely rare. Here we introduce the horse manure rolling (HMR) behavior of wild giant pandas (Ailuropoda melanoleuca). Pandas not only frequently sniffed and wallowed in fresh horse manure, but also actively rubbed the fecal matter all over their bodies. The frequency of HMR events was highly correlated with an ambient temperature lower than 15 °C. BCP/BCPO (beta-caryophyllene/caryophyllene oxide) in fresh horse manure was found to drive HMR behavior and attenuated the cold sensitivity of mice by directly targeting and inhibiting transient receptor potential melastatin 8 (TRPM8), an archetypical cold-activated ion channel of mammals. Therefore, horse manure containing BCP/BCPO likely bestows the wild giant pandas with cold tolerance at low ambient temperatures. Together, our study described an unusual behavior, identified BCP/BCPO as chemical inhibitors of TRPM8 ion channel, and provided a plausible chemistry-auxiliary mechanism, in which animals might actively seek and utilize potential chemical resources from their habitat for temperature acclimatization.

A paradigm of thermal adaptation in penguins and elephants by tuning cold activation in TRPM8.

To adapt to habitat temperature, vertebrates have developed sophisticated physiological and ecological mechanisms through evolution. Transient receptor potential melastatin 8 (TRPM8) serves as the primary sensor for cold. However, how cold activates TRPM8 and how this sensor is tuned for thermal adaptation remain largely unknown. Here we established a molecular framework of how cold is sensed in TRPM8 with a combination of patch-clamp recording, unnatural amino acid imaging, and structural modeling. We first observed that the maximum cold activation of TRPM8 in eight different vertebrates (i.e., African elephant and emperor penguin) with distinct side-chain hydrophobicity (SCH) in the pore domain (PD) is tuned to match their habitat temperature. We further showed that altering SCH for residues in the PD with solvent-accessibility changes leads to specific tuning of the cold response in TRPM8. We also observed that knockin mice expressing the penguin's TRPM8 exhibited remarkable tolerance to cold. Together, our findings suggest a paradigm of thermal adaptation in vertebrates, where the evolutionary tuning of the cold activation in the TRPM8 ion channel through altering SCH and solvent accessibility in its PD largely contributes to the setting of the cold-sensitive/tolerant phenotype.

A folding reaction at the C-terminal domain drives temperature sensing in TRPM8 channels.

In mammals, temperature-sensitive TRP channels make membrane conductance of cells extremely temperature dependent, allowing the detection of temperature ranging from noxious cold to noxious heat. We progressively deleted the distal carboxyl terminus domain (CTD) of the cold-activated melastatin receptor channel, TRPM8. We found that the enthalpy change associated with channel gating is proportional to the length of the CTD. Deletion of the last 36 amino acids of the CTD transforms TRPM8 into a reduced temperature-sensitivity channel (Q10 ∼4). Exposing the intracellular domain to a denaturing agent increases the energy required to open the channel indicating that cold drives channel gating by stabilizing the folded state of the CTD. Experiments in the presence of an osmoticant agent suggest that channel gating involves a change in solute-inaccessible volume in the CTD of ∼1,900 Å3 This volume matches the void space inside the coiled coil according to the cryogenic electron microscopy structure of TRPM8. The results indicate that a folding-unfolding reaction of a specialized temperature-sensitive structure is coupled to TRPM8 gating.