thermoTRP Channels Research Articles

Anoctamin 1 Mediates Thermal Pain as a Heat Sensor

Vertebrates can sense and avoid noxious heat that evokes pain. Many thermoTRP channels are associated with temperature sensation. TRPV1 is a representative ion channel that is activated by noxious heat. Anoctamin 1 (ANO1) is a Cl- channel activated by calcium that is highly expressed in small sensory neurons, colocalized with markers for nociceptors, and most surprisingly, activated by noxious heat over 44oC. Although ANO1 is a Cl- channel, opening of this channel leads to depolarization of sensory neurons, suggesting a role in nociception. Indeed, the functional deletion of ANO1 in sensory neurons triggers the reduction in thermal pain sensation. Thus, it seems clear that ANO1 is a heat sensor in a nociceptive pathway. Since ANO1 modulators are developed for the purpose of treating chronic diseases such as cystic fibrosis, this finding is likely to predict unwanted effects and provide a guide for better developmental strategy

PW02-035 - A role for thermo-TRP channels in innate immunity?

Exposure to cold can induce an exaggerated (local and systemic) inflammatory response in a number of rare disorders, including cryopyrin-associated periodic syndrome (CAPS), and idiopathic cold urticaria (CU). Although it is widely recognized that temperature sensing in neurons is mediated by several transient receptor potential (TRP) channels, it is not known how immune cells sense cold temperatures.

Behavioral and Electrophysiological Study of Thermal and Mechanical Pain Modulation by TRP Channel Agonists

Transient receptor potential channels (TRP) have been extensively investigated over the past few years. Recent findings in the field of pain have established a family of six thermoTRP channels (TRPA1, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4) that exhibits sensitivity to increases or decreases in temperature, as well as to chemical substances eliciting the respective hot or cold sensations. Such irritants include menthol, cinnamaldehyde, gingerol, mustard oil, capsaicin, camphor, eugenol, and others. In this study, we used behavioral and electrophysiological methods to investigate if mustard oil (allyl isothiocyanate, AITC) and capsaicin affect the sensitivity to thermal, innocuous cold, and mechanical stimuli in male rats. Unilateral intraplantar injections of AITC and capsaicin induced significant decreases in the latency for ipsilateral paw withdrawal from a noxious heat stimulus, i.e., heat hyperalgesia. These agents also significantly reduced the mechanical withdrawal thresholds of the injected paw, i.e., mechanical allodynia. Bilateral intraplantar injections of AITC resulted in a twophase effect on cold avoidance (thermal preference test). A low concentration of AITC (5%) did not change cold avoidance similarly to the vehicle control, while higher AITC concentrations (10 and 15%) significantly reduced cold avoidance, i.e., induced cold hypoalgesia. Capsaicin acted in almost the same manner. These results indicate that TRPA1 channels are clearly involved in pain reactions, and the TRPA1 agonist AITC enhances the heat pain sensitivity, possibly by indirectly modulating TRPV1 channels co-expressed in nociceptors with TRPA1s. In electrophysiological experiments, neuronal responses to electrical and graded mechanical and noxious thermal stimulations were tested before and after cutaneous application of AITC. Repetitive application of AITC initially increased the firing rate of spinal wide-dynamic range neurons; this was followed by rapid desensitization that persisted when AITC application was reapplied 30 min later. The responses to noxious thermal (but not to mechanical) stimuli were significantly enhanced irrespective of whether the neuron was directly activated by AITC. These findings indicate that AITC produced peripheral sensitization of heat nociceptors. Overall, our data support the role of hermosensitive TRPA1 and TRPV1 channels in pain modulation and show that these thermoTRP channels are promising targets for the development of a new group of analgesic drugs.

The Role of Allosteric Coupling on Thermal Activation of Thermo-TRP Channels

Thermo-transient receptor potential channels display outstanding temperature sensitivity and can be directly gated by low or high temperature, giving rise to cold- and heat-activated currents. These constitute the molecular basis for the detection of changes in ambient temperature by sensory neurons in animals. The mechanism that underlies the temperature sensitivity in thermo-transient receptor potential channels remains unknown, but has been associated with large changes in standard-state enthalpy (ΔHo) and entropy (ΔSo) upon channel gating. The magnitude, sign, and temperature dependence of ΔHo and ΔSo, the last given by an associated change in heat capacity (ΔCp), can determine a channel’s temperature sensitivity and whether it is activated by cooling, heating, or both, if ΔCp makes an important contribution. We show that in the presence of allosteric gating, other parameters, besides ΔHo and ΔSo, including the gating equilibrium constant, the strength- and temperature dependence of the coupling between gating and the temperature-sensitive transitions, as well as the ΔHo/ΔSo ratio associated with them, can also determine a channel’s temperature-dependent activity, and even give rise to channels that respond to both cooling and heating in a ΔCp-independent manner.

TRP Channels in Dental Pain

Despite the high incidence of dental pain, the mechanism underlying its generation is mostly unknown. Functional expression of temperature-sensitive transient receptor potential (thermo-TRP) channels, such as TRPV1, TRPV2, TRPM8, and TRPA1 in dental primary afferent neurons and TRPV1, TRPV2, TRPV3, TRPV4, and TRPM3 in odontoblasts, has been demonstrated and suggested as responsible for dental pain elicited by hot and cold food. However, dental pain induced by light touch or sweet substance cannot be explained by the role of thermo-TRP channels. Most of current therapeutics of dentin hypersensitivity is based on hydrodynamic theory, which argues that light stimuli such as air puff and temperature changes cause fluid movement within dentinal tubule, which is then transduced as pain. To test this theory, various TRP channels as candidates of cellular mechanotransducers were studied for expression in dental primary afferents and odontoblasts. The expression of TRPV1, TRPV2, TRPA1, TRPV4, and TRPM3 in trigeminal neurons and TRPV1, TRPV2, TRPV3, TRPV4 and TRPM3 in odontoblasts has been revealed. However, their roles as cellular mechanotransducers are controversial and contribution to generation of dental pain is still elusive. This review discusses recent advances in understanding of molecular mechanism underlying development of dental pain.

Temperature Integration at the AC Thermosensory Neurons inDrosophila

Temperature sensation has a strong impact on animal behavior and is necessary for animals to avoid exposure to harmful temperatures. It is now well known that thermoTRP (transient receptor potential) channels in thermosensory neurons detect a variable range of temperature stimuli. However, little is known about how a range of temperature information is relayed and integrated in the neural circuits. Here, we show novel temperature integration between two warm inputs via Drosophila TRPA channels, TRPA1 and Pyrexia (Pyx). The internal AC (anterior cell) thermosensory neurons, which express TRPA1, detect warm temperatures and mediate temperature preference behavior. We found that the AC neurons were activated twice when subjected to increasing temperatures. The first response was at ∼25°C via TRPA1 channel, which is expressed in the AC neurons. The second response was at ∼27°C via the second antennal segments, indicating that the second antennal segments are involved in the detection of warm temperatures. Further analysis reveals that pyx-Gal4-expressing neurons have synapses on the AC neurons and that mutation of pyx eliminates the second response of the AC neurons. These data suggest that AC neurons integrate both their own TRPA1-dependent temperature responses and a Pyx-dependent temperature response from the second antennal segments. Our data reveal the first identification of temperature integration, which combines warm temperature information from peripheral to central neurons and provides the possibility that temperature integration is involved in the plasticity of behavioral outputs.

Screening for TRPV1 Temperature-Sensing Domains with Peptide Insertion

As the prototype of temperature-activated thermoTRP channels, the vanilloid transient receptor potential channel TRPV1 is involved in acute noxious thermosensation and thermal hyperalgesia. Its temperature sensing mechanism remains mysterious and controversial. Multiple heat-sensing domains that spread across the protein have been proposed, such as the intracellular N-terminal segment between the ankyrin repeats domain and S1, the outer pore region, and the intracellular C terminus. To test the contribution of various channel regions to heat activation, we introduced short peptide insertions to uncouple peripheral domains from the pore-forming core domain. Effects on heat- and agonist-induced channel activation were tested with both calcium imaging and electrophysiological recordings. While disruption of covalent periphery-core interaction weakened desensitization upon prolonged capsaicin application, none of the tested insertions appeared to significantly alter heat activation. Proton activation and Mg2+ activation, which are mediated by the outer pore, were also not significantly affected. These observations are consistent with the transmembrane core domain being intrinsically temperature-sensitive.

Roles of Glia, Immune Cells and the Thermo-TRP Channels, TRPV1, TRPA1 and TRPM8, in Pathological Pain

Studies into the interactions between glia/immune cells and neurons have focused on the induction of patho-logical (neuropathic or inflammatory) pain. Growing evidence of close relationships between peripheral and central glia and pathological pain has emerged during the last 2 decades. Numerous experimental studies have showed the release of cytokines and inflammatory neuropeptides from peripheral and central terminals of primary sensory neurons and from ac-tivated peripheral and central glia after nerve injury (crush, ligation or transection), which in turn act in a paracrine or autocrine manner. Cytokines induce the synthesis of algogens (pain-inducing substances such as prostaglandin) which leads to the primary (peripheral) or secondary (central) sensitization responsible for hyperalgesia or allodynia under in-flammatory conditions. The review has also highlighted the role of thermo transient receptor potential (TRP) channels TRPV1, TRPA1 and TRPM8 in the induction of pathological pain. The noxious heat sensor TRPV1 has an overt role in noxious heat hyperalgesia or allodynia, whereas TRPA1 and TRPM8 seem to have roles in noxious cold or mechanical al-lodynia, although results are inconsistent. Close mutual interrelationships between immune and glial cells and thermoTRP channels via cytokines or pro-inflammatory neuropeptides cannot be ignored when attempting to explain the induction and continuation of pathological pain. Investigations on the initial signals sent to the central area (superficial dorsal horn) remote from injured (or infectious) sites are a key point to clarify the mechanisms of pathological pain.

The thermo‐TRP ion channel family: properties and therapeutic implications

The thermo-transient receptor potentials (TRPs), a recently discovered family of ion channels activated by temperature, are expressed in primary sensory nerve terminals where they provide information about thermal changes in the environment. Six thermo-TRPs have been characterised to date: TRP vanilloid (TRPV) 1 and 2 are activated by painful levels of heat, TRPV3 and 4 respond to non-painful warmth, TRP melastatin 8 is activated by non-painful cool temperatures, while TRP ankyrin (TRPA) 1 is activated by painful cold. The thermal thresholds of many thermo-TRPs are known to be modulated by extracellular mediators, released by tissue damage or inflammation, such as bradykinin, PG and growth factors. There have been intensive efforts recently to develop antagonists of thermo-TRP channels, particularly of the noxious thermal sensors TRPV1 and TRPA1. Blockers of these channels are likely to have therapeutic uses as novel analgesics, but may also cause unacceptable side effects. Controlling the modulation of thermo-TRPs by inflammatory mediators may be a useful alternative strategy in developing novel analgesics.

New Strategies to Develop Novel Pain Therapies: Addressing Thermoreceptors from Different Points of View

One approach to develop successful pain therapies is the modulation of dysfunctional ion channels that contribute to the detection of thermal, mechanical and chemical painful stimuli. These ion channels, known as thermoTRPs, promote the sensitization and activation of primary sensory neurons known as nociceptors. Pharmacological blockade and genetic deletion of thermoTRP have validated these channels as therapeutic targets for pain intervention. Several thermoTRP modulators have progressed towards clinical development, although most failed because of the appearance of unpredicted side effects. Thus, there is yet a need to develop novel channel modulators with improved therapeutic index. Here, we review the current state-of-the art and illustrate new pharmacological paradigms based on TRPV1 that include: (i) the identification of activity-dependent modulators of this thermoTRP channel; (ii) the design of allosteric modulators that interfere with protein-protein interaction involved in the functional coupling of stimulus sensing and gate opening; and (iii) the development of compounds that abrogate the inflammation-mediated increase of receptor expression in the neuronal surface. These new sites of action represent novel strategies to modulate pathologically active TRPV1, while minimizing an effect on the TRPV1 subpopulation involved in physiological and protective roles, thus increasing their potential therapeutic use.

Membrane‐tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC(50)<10 μM), without significantly affecting other thermoTRP channels. In contrast, its retrosequence or the corresponding sequences of other TRPV channels did not alter TRPV1 channel activity (IC(50)>100 μM). TRP-p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP-p5 interferes with protein-protein interactions at the level of the TRP domain that are essential for the "conformational" change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage-independent, sequence-specific TRPV1 blockers. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.

The Pore Turret of ThermoTRP Channels is a Portable Domain Contributing to Temperature Sensing

ThermoTRP channels sense ambient temperature and function as thermosensor, however, the mechanism for sensitive temperature-dependent activation remains unknown. Recent studies point to the outer pore region as an important gating structure. Our previous study shows pore turret moves substantially during temperature-driven activation, which isn’t seen during capsaicin-driven activation. Present research focuses on contribution of pore turret to heat-induced activation using a structural perturbation approach. Partial deletions and sequence replacements are performed with TRPV1 turret. Additionally, the turret is swamped between TRPV1 and TRPV2/TRPV3. All mutant channels exhibit similar capsaicin sensitivity to wildtype TRPV1 with a less than 10-fold shift in EC50, which is consistent with the notion that temperature and ligand activate TRPV1 through different pathways. In contrast, deletion and replacement mutants exhibit activation at a very different temperature, show rapid inactivation during heating, or lose temperature response. Interestingly, TRPV1 chimera containing TRPV3 turret V1/3S behaves like wildtype TRPV3 and activates at a much lower temperature, while TRPV1 chimera containing TRPV2 turret V1/2S activates at a much higher temperature like wildtype TRPV2. These results reveal that pore turret is a portable domain contributing to temperature sensing.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Extracellular Cation Gates TRPV1 via the Heat Activation Pathway

ThermoTRP channels are expressed in sensory neurons where they detect a variety of physical and chemical stimuli including temperature, ionic strength, membrane voltage and ligand binding. However, the precise mechanisms underlying such diverse modes of activation remain largely unknown. It is found that temperature and capsaicin activate TRPV1 through separate pathways. Here we investigate how extracellular cation activates the heat-sensitive TRPV1 channel.At room temperature, Mg++ ions effectively and selectively activate TRPV1 from the extracellular side in a dose-dependent manner. The relative open probability induced by 100 mM [Mg++]out is approximately 40% of that elicited by saturating capsaicin, while the same concentration of [Mg++]in is ineffective. Mg++ substantially shifts the temperature sensitivity of the channel. At 10 mM, Mg++ shifts the takeoff temperature of TRPV1 greatly, which implies that the effects of heat and Mg++ are coupled tightly. Furthermore, mutant channels with an artificial pore turret, which have less temperature sensitivity but maintain normal capsaicin responses, also show reduced cation responses. In summary, our observations suggest that in TRPV1 the pore turret may detect both heat and ion strength, and effectively transfer the energy to open the activation gate.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Current perspectives on the modulation of thermo-TRP channels: new advances and therapeutic implications

The thermo transient receptor potential (TRP) ion channels, a recently discovered family of ion channels activated by temperature, are expressed in primary sensory nerve terminals, where they provide information regarding thermal changes in the environment. Six thermo-TRPs have been characterized to date: TRPV1–4, which respond to different levels of warmth and heat, and TRPM8 and TRPA1, which respond to cool temperatures. We review the current state of knowledge of thermo-TRPs, and of the modulation of their thermal thresholds by a range of inflammatory mediators. Blockers of these channels are likely to have therapeutic uses as novel analgesics but may also cause unacceptable side effects. Controlling the modulation of thermo-TRPs by inflammatory mediators may be a useful alternative strategy in developing novel analgesics.

Characterization Of Selective TRPM8 Ligands And Their Structure Activity Response (S.A.R) Relationship

Transient receptor potential melastatin-8 (TRPM8) is an ion channel expressed extensively in sensory nerves, human prostate and overexpressed in a variety of cancers including prostate, breast, lung, colon and skin melanomas. It is activated by innoxious cooling and chemical stimuli. TRPM8 activation by cooling or chemical agonists is reported to induce profound analgesia in neuropathic pain conditions. Known TRPM8 agonists like menthol and icilin cross-activate other thermo-TRP channels like TRPV3 and TRPA1 and mutually inhibit TRPM8. This limits the usefulness of menthol and icilin as TRPM8 ligands. Consequently, the identification of selective and potent ligands for TRPM8 is of high relevance both in basic research and for therapeutic applications. In the present investigation, a group of menthol derivates was characterized. These ligands are selective and potent agonists of TRPM8. Interestingly they do not activate other thermo-TRPs like TRPA1, TRPV1, TRPV2, TRPV3 and TRPV4. These ion channels are also nociceptors and target of many inflammatory mediators. Investigations were performed in a recombinant system: Xenopus oocytes microinjected with cRNA of gene of interest were superfused with the test substances after initial responses of known standard agonists. Evoked currents were measured by two-electrode voltage clamp technique. The newly characterized ligands possess an up to six-fold higher potency (EC50 in low microM) and an up to two-fold increase in efficacy compared to the parent compound menthol. In addition, it is found that chemical derivatives of menthol like CPS-368, CPS-369, CPS-125, WS-5 and WS-12 are the most selective ligands for TRPM8. The enhanced activity and selectivity seems to be conferred by hexacyclic ring structure present in all ligands as substances like WS-23 which lack this functional group activate TRPM8 with much lower potency (EC50 in mM) and those with pentacyclcic ring structure (furanone compounds) are totally inactive. The new substances activate TRPM8 with a higher potency, efficacy and specificity than menthol and will thus be of importance for the development of pharmacological agents suitable for treatment and diagnosis of certain cancers and as analgesics. STATEMENT OF NOVELTY: The new compounds have an unmatched specificity for TRPM8 ion channels with additional display of high potency and efficacy. Thus these substances are better pharmacological tools for TRPM8 characterization then known compounds and it is suggested that these menthol-derivates may serve as model substances for the development of TRPM8 ligands.

Reply to Yao et al.: Is the pore turret just thermoTRP channels’ appendix?

Numerous studies of thermosensitive transient receptor potential (thermoTRP) channels have focused on the outer pore that contains sites critical for the channel's sensitivity to temperature, acidification, spider toxin, and other gating modulators (Fig. 1). Two segments of each subunit contribute to the outer pore structure: one is between S5 and the pore helix, the other is between the selectivity filter and S6. For the convenience of discussion, we call them the large turret and the small turret, respectively. From the available structural information of tetrameric channels, it is expected that the two turrets interact extensively. Indeed, the sites affecting gating spread across the outer pore landscape. Of particular interests are a number of sites that when mutated, lead to permanent opening or closing of the temperature-activation gate (1–3).

Topical application of l-menthol induces heat analgesia, mechanical allodynia, and a biphasic effect on cold sensitivity in rats

Menthol is used in analgesic balms and also in foods and oral hygiene products for its fresh cooling sensation. Menthol enhances cooling by interacting with the cold-sensitive thermoTRP channel TRPM8, but its effect on pain is less well understood. We presently used behavioral methods to investigate effects of topical menthol on thermal (hot and cold) pain and innocuous cold and mechanical sensitivity in rats. Menthol dose-dependently increased the latency for noxious heat-evoked withdrawal of the treated hindpaw with a weak mirror-image effect, indicating antinociception. Menthol at the highest concentration (40%) reduced mechanical withdrawal thresholds, with no effect at lower concentrations. Menthol had a biphasic effect on cold avoidance. At high concentrations (10% and 40%) menthol reduced avoidance of colder temperatures (15 °C and 20 °C) compared to 30 °C, while at lower concentrations (0.01–1%) menthol enhanced cold avoidance. In a −5 °C cold plate test, 40% menthol significantly increased the nocifensive response latency (cold hypoalgesia) while lower concentrations were not different from vehicle controls. These results are generally consistent with neurophysiological and human psychophysical data and support TRPM8 as a potential peripheral target of pain modulation.

TRPV2 Enhances Axon Outgrowth through Its Activation by Membrane Stretch in Developing Sensory and Motor Neurons

Thermosensitive TRP (thermo TRP) channels are well recognized for their contributions to sensory transduction, responding to a wide variety of stimuli including temperature, nociceptive stimuli, touch, and osmolarity. However, the precise roles for the thermo TRP channels during development have not been determined. To explore the functional importance of thermo TRP channels during neural development, the temporal expression was determined in embryonic mice. Interestingly, TRPV2 expression was detected in spinal motor neurons in addition to the dorsal root ganglia from embryonic day 10.5 and was localized in axon shafts and growth cones, suggesting that the channel is important for axon outgrowth regulation. We revealed that endogenous TRPV2 was activated in a membrane stretch-dependent manner in developing neurons by knocking down the TRPV2 function with dominant-negative TRPV2 and TRPV2-specific shRNA and significantly promoted axon outgrowth. Thus, for the first time we revealed that TRPV2 is an important regulator for axon outgrowth through its activation by membrane stretch during development.

Thermosensitive TRP channel pore turret is part of the temperature activation pathway

Temperature sensing is crucial for homeotherms, including human beings, to maintain a stable body core temperature and respond to the ambient environment. A group of exquisitely temperature-sensitive transient receptor potential channels, termed thermoTRPs, serve as cellular temperature sensors. How thermoTRPs convert thermal energy (heat) into protein conformational changes leading to channel opening remains unknown. Here we demonstrate that the pathway for temperature-dependent activation is distinct from those for ligand- and voltage-dependent activation and involves the pore turret. We found that mutant channels with an artificial pore turret sequence lose temperature sensitivity but maintain normal ligand responses. Using site-directed fluorescence recordings we observed that temperature change induces a significant rearrangement of TRPV1 pore turret that is coupled to channel opening. This movement is specifically associated to temperature-dependent activation and is not observed during ligand- and voltage-dependent channel activation. These observations suggest that the turret is part of the temperature-sensing apparatus in thermoTRP channels, and its conformational change may give rise to the large entropy that defines high temperature sensitivity.

Acute and chronic effects of neurotrophic factors BDNF and GDNF on responses mediated by thermo-sensitive TRP channels in cultured rat dorsal root ganglion neurons

Neurotrophic factors (NTFs), beside regulating neuronal survival in the central and peripheral nervous system, are also involved in the modulation of neuronal function in the adult animal. Both brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) levels are altered in pathological pain states, and exogenous BDNF and GDNF have multiple effects on pain behavior, depending on the animal model (i.e. inflammatory vs. neuropathic). Thermally gated TRP channels TRPM8, TRPA1 and TRPV1 play a significant role in pain signaling and their pattern and level of expression as well as their biophysical properties are altered in chronic pain states. Our aim was to investigate the effect of long-term and acute exposure to BDNF and GDNF on the functional expression of these thermoTRP channels in cultured rat dorsal root ganglion (DRG) neurons. We found that while BDNF treatment primarily increased the fraction of capsaicin-sensitive (TRPV1-expressing) neurons, GDNF exposure led to an increase in the allyl isothiocyanate (AITC)-responding (TRPA1-expressing) population. Moreover, BDNF treatment increased the amplitude of the response to both AITC and capsaicin. Acute treatment with both NTFs leads to a reduction in the magnitude of tachyphylaxis to noxious stimuli (heat and AITC). Overall, our data provides evidence for a role of BDNF and GDNF in regulating the pattern of expression and level of activity of the transducer channels TRPA1 and TRPV1, leading to enhanced neuronal sensitivity to painful stimuli and increased co-expression of thermoTRP channels.