Menthol Receptor TRPM8 Research Articles

Mechanisms of sensory adaptation and inhibition of the cold and menthol receptor TRPM8.

Our sensory adaptation to cold and chemically induced coolness is mediated by the intrinsic property of TRPM8 channels to desensitize. TRPM8 is also implicated in cold-evoked pain disorders and migraine, highlighting its inhibitors as an avenue for pain relief. Despite the importance, the mechanisms of TRPM8 desensitization and inhibition remained unclear. We found, using cryo-electron microscopy, electrophysiology, and molecular dynamics simulations, that TRPM8 inhibitors bind selectively to the desensitized state of the channel. These inhibitors were used to reveal the overlapping mechanisms of desensitization and inhibition and that cold and cooling agonists share a common desensitization pathway. Furthermore, we identified the structural determinants crucial for the conformational change in TRPM8 desensitization. Our study illustrates how receptor-level conformational changes alter cold sensation, providing insights into therapeutic development.

The Cold Receptor TRPM8 Mediates the Antioxidant and Cytoprotective Effects of Mild Hypothermia in Cerebral Endothelial Cells During Inflammation

Cerebral vascular endothelium, the key component of the neurovascular unit, is most vulnerable to oxidative stress injury. Our previous reports in newborn pigs indicated that brain hypothermia prevents cerebral endothelial injury by oxidative stress caused by neonatal seizures and asphyxia. We used primary cerebral vascular endothelial cells (CVEC) from newborn pigs as an in vitro model of oxidative stress condition in the brain caused by inflammation. During normothermia (37˚C), CVEC respond to a pro‐inflammatory cytokine TNF‐alpha (30 ng/ml) by excessive formation of reactive oxygen species (ROS) leading to cell death by apoptosis (caspase‐3 activation, DNA fragmentation, loss of cell‐cell contacts). Remarkably, mild hypothermia (24.5°C) reduced ROS and prevented apoptosis in CVEC exposed to TNF‐alpha. We tested the hypothesis that the cold sensing menthol receptor TRPM8 mediates the cytoprotective effects of mild hypothermia in cerebral endothelium. TRPM8 (mRNA and protein) is expressed in CVEC. The TRPM8 agonist icilin (5 µM) reduced ROS and prevented apoptosis in CVEC exposed to TNF‐alpha at 37˚C, thus mimicking the cytoprotective effects of mild cooling. The TRPM8 antagonist AMTB (20 µM) reversed the icilin‐mediated cytoprotective effects during inflammation. The cell surface biotinylation technique suggested that mild hypothermia and icilin (at 37˚C) promote a time‐dependent TRPM8 translocation from an intracellular compartment to the plasma membrane concomitantly with the receptor activation. We used the in vitro model of the blood‐brain barrier (BBB) to test the functional consequences of TRPM8‐mediated cytoprotection. Confluent monolayers of CVEC cultured on transwell inserts with semipermeable membranes were exposed to TNF‐alpha during normothermia and mild hypothermia conditions. The BBB properties were detected by transendothelial electrical resistance (TEER) and the permeability to 3 kDa fluorescent Dextran. During a 2‐6 h exposure to TNF‐alpha at 37°C, TEER was time‐dependently reduced, and the Dextran permeability was elevated indicating the BBB opening. Mild hypothermia (24.5°C) and icilin (at 37˚C) largely preserved the BBB properties during inflammatory conditions. Overall, our findings demonstrate that mild hypothermia, via a TRPM8‐mediated mechanism, reduces oxidative stress, promotes cell survival, and prevents BBB damage during inflammation. The TRPM8 targeting approaches may provide a promising therapeutic strategy for cerebrovascular disease caused by brain inflammation.

Activation of the Cold and Menthol Receptor TRPM8 Improves Post-Traumatic Recovery of Rat Muscle Soleus During Fullerene Treatment

Activation of the Cold and Menthol Receptor TRPM8 Improves Post-Traumatic Recovery of Rat Muscle Soleus During Fullerene Treatment

Evolution of the human cold/menthol receptor, TRPM8

Genes showing versatile functions or subjected to fast expansion and contraction during the adaptation of species to specific ecological conditions, like sensory receptors for odors, pheromones and tastes, are characterized by a great plasticity through evolution. One of the most fascinating sensory receptors in the family of TRP channels, the cold and menthol receptor TRPM8, has received significant attention in the literature. Recent studies have reported the existence of TRPM8 channel isoforms encoded by alternative mRNAs transcribed from alternative promoters and processed by alternative splicing. Since the first draft of the human genome was accomplished in 2000, alternative transcription, alternative splicing and alternative translation have appeared as major sources of gene product diversity and are thought to participate in the generation of complexity in higher organisms. In this study, we investigate whether alternative transcription has been a driving force in the evolution of the human forms of the cold receptor TRPM8.We identified 33 TRPM8 alternative mRNAs (24 new sequences) and their associated protein isoforms in human tissues. Using comparative genomics, we described the evolution of the human TRPM8 sequences in eight ancestors since the origin of Amniota, and estimated in which ancestors the new TRPM8 variants originated. In order to validate the estimated origins of this receptor, we performed experimental validations of predicted exons in mouse tissues. Our results suggest a first diversification event of the cold receptor in the Boreoeutheria ancestor, and a subsequent divergence at the origin of Simiiformes.

Selective cold pain inhibition by targeted block of TRPM8-expressing neurons with quaternary lidocaine derivative QX-314

Treatment of pain with local anesthetics leads to an unfavorable decrease in general sensory acuity due to their indiscriminate block of both pain sensing (nociceptors) and non-pain sensing nerves. However, the cell impermeant lidocaine derivative QX-314 can be selectively targeted to only nociceptors by permeation through ligand-gated cation channels. Here we show that localized injection of QX-314 with agonists for the menthol receptor TRPM8 specifically blocks cold-evoked behaviors in mice, including cold allodynia and hyperalgesia. Remarkably, cooling stimuli also promotes QX-314-mediated inhibition of cold behaviors, and can be used to block cold allodynia, while retaining relatively normal cold sensation. The effects of both agonist and thermally evoked uptake of QX-314 are TRPM8-dependent, results demonstrating an effective approach to treat localized cold pain without altering general somatosensation.

TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells.

The cold and menthol receptor TRPM8 is highly expressed in prostate and prostate cancer (PC). Recently, we identified that TRPM8 is as an ionotropic testosterone receptor. The TRPM8 mRNA is expressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Here, from the chromatin-immunoprecipitation (ChIP) analysis, we found that an androgen response element (ARE) mediates androgen regulation of trpm8. Furthermore, using immunofluorescence, calcium-imaging and planar lipid bilayers, we identified that TRPM8 channel is functionally regulated by androgens in the prostate. Although TRPM8 mRNA is expressed at high levels, we found that the TRPM8 protein undergoes ubiquitination and degradation in PC cells. The mass-spectrometry analysis of TRPM8, immunoprecipitated from LNCaP cells identified ubiquitin-like modifier-activating enzyme 1 (UBA1). PYR-41, a potent inhibitor of initial enzyme in the ubiquitination cascade, UBA1, increased TRPM8 activity on the plasma membrane (PM) of LNCaP cells. Furthermore, PYR-41-mediated PMTRPM8 activity was accompanied by enhanced activation of p53 and Caspase-9. Interestingly, we found that the trpm8 promoter possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. In addition to the genomic regulation of TRPM8 by AR and p53, our findings indicate that the testosterone-induced PMTRPM8 activity elicits Ca2+ uptake, subsequently causing apoptotic cell death. These findings support the strategy of rescuing PMTRPM8 expression as a new therapeutic application through the regulation of PC cell growth and proliferation.

Artemin, a Glial Cell Line-Derived Neurotrophic Factor Family Member, Induces TRPM8-Dependent Cold Pain

Chronic pain associated with injury or disease can result from dysfunction of sensory afferents whereby the threshold for activation of pain-sensing neurons (nociceptors) is lowered. Neurotrophic factors control nociceptor development and survival, but also induce sensitization through activation of their cognate receptors, attributable, in part, to the modulation of ion channel function. Thermal pain is mediated by channels of the transient receptor potential (TRP) family, including the cold and menthol receptor TRPM8. Although it has been shown that TRPM8 is involved in cold hypersensitivity, the molecular mechanisms underlying this pain modality are unknown. Using microarray analyses to identify mouse genes enriched in TRPM8 neurons, we found that the glial cell line-derived neurotrophic factor (GDNF) family receptor GFRα3 is expressed in a subpopulation of TRPM8 sensory neurons that have the neurochemical profile of cold nociceptors. Moreover, we found that artemin, the specific GFRα3 ligand that evokes heat hyperalgesia, robustly sensitized cold responses in a TRPM8-dependent manner in mice. In contrast, GFRα1 and GFRα2 are not coexpressed with TRPM8 and their respective ligands GDNF and neurturin did not induce cold pain, whereas they did evoke heat hyperalgesia. Nerve growth factor induced mild cold sensitization, consistent with TrkA expression in TRPM8 neurons. However, bradykinin failed to alter cold sensitivity even though its receptor expresses in a subset of TRPM8 neurons. These results show for the first time that only select neurotrophic factors induce cold sensitization through TRPM8 in vivo, unlike the broad range of proalgesic agents capable of promoting heat hyperalgesia.

The TRPM8 ion channel comprises direct Gq protein-activating capacity

The transient receptor potential (TRP) family of ion channels comprises receptors that are activated by a vast variety of physical as well as chemical stimuli. TRP channels interact in a complex manner with several intracellular signaling cascades, both up- and downstream of receptor activation. Investigating cascades stimulated downstream of the cold and menthol receptor TRPM8, we found evidence for both, functional and structural interaction of TRPM8 with Gαq. We demonstrated menthol-evoked increase in intracellular Ca(2+) under extracellular Ca(2+)-free conditions, which was blocked by the PLC inhibitors U73122 or edelfosine. This metabotropic Ca(2+) signal could be observed also in cells expressing a channel-dead (i.e. non-conducting) or a chloride-conducting TRPM8 pore mutant. However, this intracellular metabotropic Ca(2+) signal could not be detected in Gαq deficient cells or in the presence of dominant-negative GαqX. Evidence for a close spatial proximity necessary for physical interaction of TRPM8 and Gαq was provided by acceptor bleaching experiments demonstrating FRET between TRPM8-CFP and Gαq-YFP. A Gαq-YFP mobility assay (FRAP) revealed a restricted diffusion of Gαq-YFP under conditions when TRPM8 is immobilized in the plasma membrane. Moreover, a menthol-induced and TRPM8-mediated G protein activation could be demonstrated by FRET experiments monitoring the dissociation of Gαq-YFP from a Gβ/Gγ-CFP complex, and by the exchange of radioactive [(35)S]GTPγS for GDP. Our observations lead to a view that extends the operational range of the TRPM8 receptor from its function as a pure ion channel to a molecular switch with additional metabotropic capacity.

EC50 and IC50 Measurements of TRPM8 in Lipid Bilayers

Ensemble measurements of ion channel currents, made in the presence of pharmaceutical compounds, enable determination of compound activity and potency in the form of IC50 and EC50 values, and are traditionally performed with Patch Clamp. An alternative platform for ion channel study involves the formation of artificial bilayers from the contact of lipid monolayers at aqueous/oil interfaces. This technique has the potential for automation and parallel measurements. Previous work with this platform has demonstrated measurements of physiologically relevant ion channels at the single molecule level. Here we present measurements of the cold and menthol receptor TRPM8 at the single channel and ensemble level. We studied the effect of menthol and temperature on the open probability of TRPM8 and found a single-channel conductance of 64 ± 6 pS, comparable to previous work. In addition, we measured ensemble activity of TRPM8 as a function of temperature and varying PI(4,5)P2 concentration. Our apparatus enables introduction of pharmaceutical compounds to the solution surrounding the bilayer during measurement. We introduced solutions containing menthol and 2-aminoethoxydiphenyl borate (2-APB) at variable concentration to bilayers during ensemble TRPM8 measurements, enabling us to efficiently and repeatedly measure an IC50 value of 4.9 μM for 2-APB and an EC50 value of 111.8 μM for menthol, both in agreement with literature. These developments indicate that artificial bilayers may play an expanded role in future ion channel studies, including drug discovery and safety screening.

Ion channel drug potency assay with an artificial bilayer chip

The potency of pharmaceutical compounds acting on ion channels can be determined through measurements of ion channel conductance as a function of compound concentration. We have developed an artificial lipid bilayer chip for simple, fast, and high-yield measurement of ion channel conductance with simultaneous solution perfusion. Here we show the application of this chip to the measurement of the mammalian cold and menthol receptor TRPM8. Ensemble measurements of TRPM8 as a function of concentration of menthol and 2-aminoethoxydiphenyl borate (2-APB) enabled efficient determination of menthol's EC(50) (111.8 μM ± 2.4 μM) and 2-APB's IC(50) (4.9 μM ± 0.2 μM) in agreement with published values. This validation, coupled with the compatibility of this platform with automation and parallelization, indicates significant potential for large-scale pharmaceutical ion channel screening.

Ligand stoichiometry of the cold‐ and menthol‐activated channel TRPM8

Temperature-sensitive transient receptor potential (TRP) channels play a key role in somatosensation, not only as primary thermosensors but also as chemosensors for various compounds that evoke a thermal sensation. The fundamental mechanisms whereby TRP channels translate ligand binding into opening of the ion conducting pore are, however, poorly understood, and the actual number of ligands that bind to these channels is fully unknown. Here, we investigated the ligand stoichiometry of the cold sensor and menthol receptor TRPM8. Based on a detailed biophysical analysis of tandem constructs of wild-type and menthol-insensitive TRPM8 subunits, we now provide direct evidence that each channel has four independent and energetically equivalent menthol interaction sites. These results suggest a fundamentally different ligand stoichiometry in TRP channels, in comparison with other major families of ligand-gated ion channels.

A spicy family tree: TRPV1 and its thermoceptive and nociceptive lineage

In the current issue, Mishra and colleagues demonstrate that mice lacking somatosensory neurons in the TRPV1 lineage are completely insensitive to thermal stimuli of any quality or modality, including both hot and cold temperatures. TRPV1 is a heat‐gated ion channel expressed in most heat‐sensitive nociceptive neurons and is the receptor for capsaicin, the pungent ingredient in ‘hot’ chili peppers ( Caterina et al , 1997 ). In the adult mouse, TRPV1 neurons are molecularly heterogeneous, but channel expression is absent in the majority of cold‐sensitive neurons labelled with the menthol receptor TRPM8 and all presumptive mechanosensory neurons expressing the Mas‐related G protein‐coupled receptor D (Mrgprd). However, using a molecular genetic approach to label TRPV1 neurons from the inception of channel expression, the authors find that TRPV1 is a broad developmental marker for the majority of somatosensory neurons, including those expressing Mrgprd and TRPM8. Moreover, genetically targeted ablation of neurons within the TRPV1 lineage eliminates a broad range of mouse behaviours including thermosensation, thermoregulation, nociception, and pruriception, yet these animals retain normal mechanosensation. These results demonstrate that TRPV1 expression predominates embryonically, but is refined over postnatal development. Moreover, other thermosensitive and nociceptive molecules, such as TRPM8, are derived from the TRPV1 lineage even though they are not entirely co‐expressed in adults. These findings expand the role of TRPV1 and show the channel has a patriarchal role as a molecular indicator of thermal and nociceptive afferent circuits. We detect changes in our environment with specialized sensory nerve endings in the skin, many of which are activated by multiple modes of somatosensory stimuli, such as thermal and mechanical. This polymodality suggests that decoding of a specific sensory modality does not occur in the afferent, but is deciphered in higher centres such as the spinal cord or brain. However, this hypothesis has come under considerable debate with …

The development of peripheral cold neural circuits based on TRPM8 expression

Afferent nerve fibers of the somatosensory system are a molecularly diverse cell population that detects a varied range of environmental stimuli, converting these external cues ultimately into a sensory percept. Afferents mediating detection of thermal stimuli express a repertoire of temperature sensitive ion channels of the TRP family which endow these nerves with the ability to respond to the breadth of temperatures in the environment. The cold and menthol receptor TRPM8 is responsible for detection of cold and, unlike other thermosensors, detects both innocuous and noxious temperatures. How this single molecule can perform such diverse functions is currently unknown, but expression analyses in adult tissues shows that TRPM8 neurons are a molecularly diverse population and it is likely that this diversity underlies differential functionality. To determine how this phenotype is established, we examined the developmental time course of TRPM8 expression using a mouse transgenic line in which GFP expression is driven by the TRPM8 transcriptional promoter (Trpm8 GFP ). We find that Trpm8 GFP expression begins prior to embryonic day 15.5 (E15.5) after which expression reaches levels observed in adult neurons. By E18.5, central axons of Trpm8 GFP neurons reach the spinal cord dorsal horn, but anatomical localization and in vivo measurements of neural activity suggest that fully functional cold circuits are not established until after the first postnatal week. Additionally, Trpm8 GFP neurons undergo a transition in neurochemical phenotype, ultimately reaching adult expression of markers such TRPV1, CGRP, peripherin, and NF200 by postnatal day 14. Thus, based on immunochemical, anatomical and functional criteria, active cold neural circuits are fully established by the second week postnatal, thereby suggesting that important extrinsic or intrinsic mechanisms are active prior to this developmental stage.

Phosphoinositide Regulation of TRPM8 Channels in Planar Lipid Bilayers

The cold and menthol receptor TRPM8 is regulated by membrane phosphoinositides. To study the effects of lipids directly on the channel, we have reconstituted the purified TRPM8 in planar lipid bilayers. This system allows full control of the lipid composition in our experiments. The reconstituted channel was activated by menthol or cold, and its activity depended on the presence of specific phosphoinositides. In the presence of menthol, TRPM8 exhibited the highest probability of opening in the presence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] with Po ∼ 0.89 at +100 mV and Po ∼ 0.4 at −100 mV. Less channel activity was induced by phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] with Po ∼ 0.53 at +100 mV and Po ∼ 0.2 at −100 mV. Phosphatidylinositol 3,4,5-bisphosphate [PI(3,4,5) P2] resulted in irregular TRPM8 channel currents and lower open probability with Po ∼ 0.21 at +100 mV and Po ∼ 0.087 at −100 mV. Among the tested lipids the lowest TRPM8 channel activity was induced by phosphatidylinositol 4-phosphate [PI(4)P] with Po ∼ 0.12 at +100 mV and Po ∼ 0.019 at −100 mV. The lipid specificity profile in lipid bilayers is very similar to that observed in excised patches.We have also studied the activation of TRPM8 channels in lipid bilayer with cold. Cooling the system with reconstituted TRPM8 channels also required the presence of PI(4,5)P2. The main shift in the channel behavior was observed in the temperature range from 21°C to 18°C where the channel showed drastic changes in the open probability from 0.05 to 0.85 at +100 mV.These results demonstrate that the TRPM8 protein is directly activated by cold, menthol and phosphoinositides.

Ice Water Testing Reveals Hypersensitivity in Adult Rats That Experienced Neonatal Bladder Inflammation: Implications for Painful Bladder Syndrome/Interstitial Cystitis

We determined whether clinical observations of hypersensitivity to ice water testing, that is infusion of ice-cold saline into the bladder, in patients with painful bladder syndrome/interstitial cystitis have a parallel in a rat model of bladder hypersensitivity produced by neonatal inflammation. Rat pups were anesthetized as neonates (postnatal days 14 to 16). In some pups the bladder was inflamed by intravesical zymosan treatment. As adults, the rats were re-anesthetized and tested for abdominal muscle contractions to ice water testing, measured on electromyogram. Various neonatally treated groups of rats underwent bladder re-inflammation/no re-inflammation and/or bladder distention before ice water testing. Other control rats were treated only in adulthood. Rats that underwent bladder inflammation as neonates manifested bladder hypersensitivity in adulthood, as indexed by significantly greater mean electromyogram responses during ice water testing. This bladder hypersensitivity did not require adult re-inflammation to manifest. Hypersensitivity was also observed with or without prior bladder distention, although the magnitude of electromyogram responses during ice water testing significantly correlated with the magnitude of electromyogram responses to bladder distention. Neonatally induced effects were not significantly related to estrous cycle phase. Exposure to menthol did not significantly enhance the overall magnitude of the electromyogram response to ice water testing in neonatally treated rats. Current results parallel those in a recent study showing that most patients with painful bladder syndrome/interstitial cystitis experience pain when undergoing ice water testing after previous urodynamic testing. These findings suggest that this animal model may be useful for understanding the etiology of and treatment for painful bladder syndrome/interstitial cystitis.

256 The Cold and Menthol Receptor TRPM8 Is Expressed in Epithelial Cells of the Human and Murine Gastric Antrum

256 The Cold and Menthol Receptor TRPM8 Is Expressed in Epithelial Cells of the Human and Murine Gastric Antrum

Transient Receptor Potential Channels in Sensory Neurons Are Targets of the Antimycotic Agent Clotrimazole

Clotrimazole (CLT) is a widely used drug for the topical treatment of yeast infections of skin, vagina, and mouth. Common side effects of topical CLT application include irritation and burning pain of the skin and mucous membranes. Here, we provide evidence that transient receptor potential (TRP) channels in primary sensory neurons underlie these unwanted effects of CLT. We found that clinically relevant CLT concentrations activate heterologously expressed TRPV1 and TRPA1, two TRP channels that act as receptors of irritant chemical and/or thermal stimuli in nociceptive neurons. In line herewith, CLT stimulated a subset of capsaicin-sensitive and mustard oil-sensitive trigeminal neurons, and evoked nocifensive behavior and thermal hypersensitivity with intraplantar injection in mice. Notably, CLT-induced pain behavior was suppressed by the TRPV1-antagonist BCTC [(N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide)] and absent in TRPV1-deficient mice. In addition, CLT inhibited the cold and menthol receptor TRPM8, and blocked menthol-induced responses in capsaicin- and mustard oil-insensitive trigeminal neurons. The concentration for 50% inhibition (IC50) of inward TRPM8 current was approximately 200 nM, making CLT the most potent known TRPM8 antagonist and a useful tool to discriminate between TRPM8- and TRPA1-mediated responses. Together, our results identify TRP channels in sensory neurons as molecular targets of CLT, and offer means to develop novel CLT preparations with fewer unwanted sensory side effects.

Thermoregulation: Channels that Are Cool to the Core

TRP channels are molecular sensors that endow animals with the ability to sense external temperatures. Recent studies have provided strong evidence that at least one TRP channel, TRPV1, is also essential for the maintenance of internal body temperature.

Channelling cold reception

Perception of cold and hot is one of life's essentials. Three research teams find that, when a temperature-sensing receptor is deleted in mice, the animals lose their response to a range of cold temperatures. A variety of different ion channels have been suggested to underlie the detection of cold stimuli, and the role of each of these in detecting innocuous cool versus noxious cold has been much debated. The menthol receptor TRPM8 is a strong candidate as a cold transducer, but its physiological importance has been questioned. A study of cold sensitivity in TRPM8 knockout mice has clarified matters. Mice lacking the receptor are deficient in the ability to sense cold at the cellular level, in intact nerve fibres and in whole animal (behavioural) tests. TRPM8 is clearly the primary contributor to peripheral cold sensation, with animals lacking this channel unable to discriminate between warm and cold surfaces until the temperature drops to below 10 °C.

Mice Left Out in the Cold: Commentary on the Phenotype of TRPM8-Nulls

Detection of innocuous temperatures allows an organism to select an appropriate environmental climate, while the ability to recognize noxious temperature extremes warns of impending tissue damage. For temperatures considered cold, the menthol receptor TRPM8 is activated when temperatures drop below ~26°C, thus making it an intriguing candidate as the molecular mediator of cold perception. However, confirmation of this hypothesis in vivo has eluded researchers until recently. Three independent research groups have reported that mice lacking this single gene are severely impaired in their ability to detect cold temperatures. Remarkably, these animals are deficient in many diverse aspects of cold signaling, including cool and noxious cold perception, injury-evoked sensitization to cold, and cooling-induced analgesia. These animals provide a great deal of insight into the molecular signaling pathways that participate in the detection of cold and painful stimuli.