Warm temperatures activate TRPV4 in mouse 308 keratinocytes

Mammalian survival requires constant monitoring of environmental and body temperature. Recently, several members of the transient receptor potential vanilloid (TRPV) subfamily of ion channels have been identified that can be gated by increases in temperature into the warm (TRPV3 and TRPV4) or painfully hot (TRPV1 and TRPV2) range. In rodents, TRPV3 and TRPV4 proteins have not been detected in sensory neurons but are highly expressed in skin epidermal keratinocytes. Here, we show that in response to warm temperatures (>32 degrees C), the mouse 308 keratinocyte cell line exhibits nonselective transmembrane cationic currents and Ca2+ influx. Both TRPV3 and TRPV4 are expressed in 308 cells. However, the warmth-evoked responses we observe most closely resemble those mediated by recombinant TRPV4 on the basis of their electrophysiological properties and sensitivity to osmolarity and the phorbol ester, 4alpha-phorbol-12,13-didecanoate. Together, these data support the notion that keratinocytes are capable of detecting modest temperature elevations, strongly suggest that TRPV4 participates in these responses, and define a system for the cell biological analysis of warmth transduction.     

TRPV channels as temperature sensors

The past year has seen a doubling in the number of heat-sensitive ion channels to six, and four of these channels are from the TRPV family. These channels characteristically have Q(10) values of >10 above the thermal threshold, very different from the Q(10) values of 1.5-2.0 seen in most ion channels. Cells expressing TRPV1 show similar temperature sensitivity to small capsaicin-sensitive nociceptor neurons, consistent with these neurons expressing homomers of TRPV1. A-delta fibres exhibit properties that may be explained by TRPV2 containing channels which is present in large diameter sensory neurons that do not express TRPV1. TRPV3 has a lower temperature threshold and may contribute to warm-sensitive channels together with TRPV1. Warm sensation may also be transduced by TRPV4 expressing sensory neurons and hypothalamic neurons. We can now look forward to further work defining the properties of the recombinant channels in more detail and a re-analysis of endogenous i(heat) currents in thermosensitive neurons and other cells. Data from the study of mice in which TRPV2, TRPV3 or TRPV4 have been deleted are also eagerly awaited.     

Heteromeric heat-sensitive transient receptor potential channels exhibit distinct temperature and chemical response

TRPV1 and TRPV3 are two heat-sensitive ion channels activated at distinct temperature ranges perceived by human as hot and warm, respectively. Compounds eliciting human sensations of heat or warmth can also potently activate these channels. In rodents, TRPV3 is expressed predominantly in skin keratinocytes, whereas in humans TRPV1 and TRPV3 are co-expressed in sensory neurons of dorsal root ganglia and trigeminal ganglion and are known to form heteromeric channels with distinct single channel conductances as well as sensitivities to TRPV1 activator capsaicin and inhibitor capsazepine. However, how heteromeric TRPV1/TRPV3 channels respond to heat and other stimuli remains unknown. In this study, we examined the behavior of heteromeric TRPV1/TRPV3 channels activated by heat, capsaicin, and voltage. Our results demonstrate that the heteromeric channels exhibit distinct temperature sensitivity, activation threshold, and heat-induced sensitization. Changes in gating properties apparently originate from interactions between TRPV1 and TRPV3 subunits. Our results suggest that heteromeric TRPV1/TRPV3 channels are unique heat sensors that may contribute to the fine-tuning of sensitivity to sensory inputs.     

TRPV1 and TRPA1 Mediate Peripheral Nitric Oxide-Induced Nociception in Mice

Nitric oxide (NO) can induce acute pain in humans and plays an important role in pain sensitization caused by inflammation and injury in animal models. There is evidence that NO acts both in the central nervous system via a cyclic GMP pathway and in the periphery on sensory neurons through unknown mechanisms. It has recently been suggested that TRPV1 and TRPA1, two polymodal ion channels that sense noxious stimuli impinging on peripheral nociceptors, are activated by NO in heterologous systems. Here, we investigate the relevance of this activation. We demonstrate that NO donors directly activate TRPV1 and TRPA1 in isolated inside-out patch recordings. Cultured primary sensory neurons display both TRPV1- and TRPA1-dependent responses to NO donors. BH4, an essential co-factor for NO production, causes activation of a subset of DRG neurons as assayed by calcium imaging, and this activation is at least partly dependent on nitric oxide synthase activity. We show that BH4-induced calcium influx is ablated in DRG neurons from TRPA1/TRPV1 double knockout mice, suggesting that production of endogenous levels of NO can activate these ion channels. In behavioral assays, peripheral NO-induced nociception is compromised when TRPV1 and TRPA1 are both ablated. These results provide genetic evidence that the peripheral nociceptive action of NO is mediated by both TRPV1 and TRPA1.     

N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.     

Farnesyl Pyrophosphate Is a Novel Pain-producing Molecule via Specific Activation of TRPV3

Temperature-sensitive transient receptor potential ion channels (thermoTRPs) expressed in epidermal keratinocytes and sensory afferents play an important role as peripheral pain detectors for our body. Many natural and synthetic compounds have been found to act on the thermoTRPs leading to altered nociception, but little is known about endogenous painful molecules activating TRPV3. Here, we show that farnesyl pyrophosphate (FPP), an intermediate metabolite in the mevalonate pathway, specifically activates TRPV3 among six thermoTRPs using Ca²⁺ imaging and electrophysiology with cultured keratinocytes and TRPV3-overexpressing cells. Agonistic potencies of related compounds in the FPP metabolism were ignorable. Voltage-dependence of TRPV3 was shifted by FPP, which appears to be the activation mechanism. An intraplantar injection of FPP acutely elicits nociceptive behaviors in inflamed animals, indicating that FPP is a novel endogenous pain-producing substance via TRPV3 activation. Co-culture experiments demonstrated that this FPP-evoked signal in the keratinocytes is transmitted to sensory neurons. In addition, FPP reduced TRPV3 heat threshold resulting in heightened behavioral sensitivity to noxious heat. Taken together, our data suggest that FPP is the firstly identified endogenous TRPV3 activator that causes nociception. Our results may provide useful chemical information to elucidate TRPV3 physiology and novel pain-related metabolisms.     

Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3

The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3‐transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists‐activated Ca²⁺ influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein‐protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes.     

The Xenopus tropicalis orthologue of TRPV3 is heat sensitive

Thermosensitive members of the transient receptor potential (TRP) family of ion channels (thermal TRP channels) play a crucial role in mammalian temperature sensing. Orthologues of these channels are present in lower vertebrates and, remarkably, some thermal TRP orthologues from different species appear to mediate opposing responses to temperature. For example, whereas the mammalian TRPV3 channel is activated by heat, frog TRPV3 is reportedly activated by cold. Intrigued by the potential implications of these opposing responses to temperature for the mechanism of temperature-dependent gating, we cloned Xenopus laevis TRPV3 and functionally expressed it in both mammalian cell lines and Xenopus oocytes. We found that, when expressed in mammalian cells, the recombinant channel lacks the reported cold sensitivity; rather, it is activated by temperatures >50°C. Furthermore, when expressed in mammalian cells, the frog orthologue shows other features characteristic of mammalian TRPV3, including activation by the agonist 2-aminoethoxydiphenyl borate and an increased response with repeated stimulation. We detected both heat- and cold-activated currents in Xenopus oocytes expressing the recombinant frog TRPV3 channel. However, cold-activated currents were also apparent in control oocytes lacking recombinant TRPV3. Our data indicate that frog TRPV3 resembles its mammalian orthologues in terms of its thermosensitivity and is intrinsically activated by heat. Thus, all known vanilloid receptors are activated by heat. Our data also show that Xenopus oocytes contain endogenous receptors that are activated by cold, and suggest that cold sensitivity of TRP channels established using Xenopus oocytes as a functional expression system may need to be revisited.     

The TRPV3 mutation associated with the hairless phenotype in rodents is constitutively active

TRPV3 is a non-selective cation channel activated by warm to hot temperatures. In rodents, TRPV3 is highly expressed in basal keratinocytes of skin and oral/nasal epithelia. TRPV3 knockout mice showed impaired responses to innocuous and noxious heat but otherwise normal appearance and reactions to many sensory modalities. However, point mutations of TRPV3 at Gly573 to Ser and Cys have recently been linked to autosomal dominant hairless phenotypes and spontaneous dermatitis in mice and rats, implicating an important role for TRPV3 in alopecia and skin diseases. Exactly, how the mutations affect TRPV3 function was unexplained. Here, we show that both G573S and G573C mutations of murine TRPV3 are constitutively active in heterologous systems. In HEK 293 cells, expression of the TRPV3 mutants causes cell death. In Xenopus oocytes, the constitutively active mutant channel is irresponsive to thermal and chemical stimuli but it reduces the temperature threshold and enhances the responses to heat and TRPV3 agonists of the wild type channel when they are co-expressed. We conclude that the G573S and G573C substitutions render the TRPV3 channel spontaneously active under normal physiological conditions, which in turn alters ion homeostasis and membrane potentials of skin keratinocytes, leading to hair loss and dermatitis-like skin diseases.     

Expression of TRPV1 Channels after Nerve Injury Provides an Essential Delivery Tool for Neuropathic Pain Attenuation

Increased expression of the transient receptor potential vanilloid 1 (TRPV1) channels, following nerve injury, may facilitate the entry of QX-314 into nociceptive neurons in order to achieve effective and selective pain relief. In this study we hypothesized that the level of QX-314/capsaicin (QX-CAP) - induced blockade of nocifensive behavior could be used as an indirect in-vivo measurement of functional expression of TRPV1 channels. We used the QX-CAP combination to monitor the functional expression of TRPV1 in regenerated neurons after inferior alveolar nerve (IAN) transection in rats. We evaluated the effect of this combination on pain threshold at different time points after IAN transection by analyzing the escape thresholds to mechanical stimulation of lateral mental skin. At 2 weeks after IAN transection, there was no QX-CAP mediated block of mechanical hyperalgesia, implying that there was no functional expression of TRPV1 channels. These results were confirmed immunohistochemically by staining of regenerated trigeminal ganglion (TG) neurons. This suggests that TRPV1 channel expression is an essential necessity for the QX-CAP mediated blockade. Furthermore, we show that 3 and 4 weeks after IAN transection, application of QX-CAP produced a gradual increase in escape threshold, which paralleled the increased levels of TRPV1 channels that were detected in regenerated TG neurons. Immunohistochemical analysis also revealed that non-myelinated neurons regenerated slowly compared to myelinated neurons following IAN transection. We also show that TRPV1 expression shifted towards myelinated neurons. Our findings suggest that nerve injury modulates the TRPV1 expression pattern in regenerated neurons and that the effectiveness of QX-CAP induced blockade depends on the availability of functional TRPV1 receptors in regenerated neurons. The results of this study also suggest that the QX-CAP based approach can be used as a new behavioral tool to detect dynamic changes in TRPV1 expression, in various pathological conditions.     

P2Y1 Receptor Activation of the TRPV4 Ion Channel Enhances Purinergic Signaling in Satellite Glial Cells

Transient receptor potential (TRP) ion channels of peripheral sensory pathways are important mediators of pain, itch, and neurogenic inflammation. They are expressed by primary sensory neurons and by glial cells in the central nervous system, but their expression and function in satellite glial cells (SGCs) of sensory ganglia have not been explored. SGCs tightly ensheath neurons of sensory ganglia and can regulate neuronal excitability in pain and inflammatory states. Using a modified dissociation protocol, we isolated neurons with attached SGCs from dorsal root ganglia of mice. SGCs, which were identified by expression of immunoreactive Kir4.1 and glutamine synthetase, were closely associated with neurons, identified using the pan-neuronal marker NeuN. A subpopulation of SGCs expressed immunoreactive TRP vanilloid 4 (TRPV4) and responded to the TRPV4-selective agonist GSK1016790A by an influx of Ca²⁺ ions. SGCs did not express functional TRPV1, TRPV3, or TRP ankyrin 1 channels. Responses to GSK1016790A were abolished by the TRPV4 antagonist HC067047 and were absent in SGCs from Trpv4^−/− mice. The P2Y₁-selective agonist 2-methylthio-ADP increased [Ca²⁺]_i in SGCs, and responses were prevented by the P2Y1-selective antagonist MRS2500. P2Y₁ receptor-mediated responses were enhanced in TRPV4-expressing SGCs and HEK293 cells, suggesting that P2Y₁ couples to and activates TRPV4. PKC inhibitors prevented P2Y₁ receptor activation of TRPV4. Our results provide the first evidence for expression of TRPV4 in SGCs and demonstrate that TRPV4 is a purinergic receptor-operated channel in SGCs of sensory ganglia.     

2-aminoethoxydiphenyl borate stimulates pulmonary C neurons via the activation of TRPV channels

This study was carried out to determine the effect of 2-aminoethoxydiphenyl borate (2-APB), a common activator of transient receptor potential vanilloid (TRPV) type 1, 2, and 3 channels, on cardiorespiratory reflexes, pulmonary C fiber afferents, and isolated pulmonary capsaicin-sensitive neurons. In anesthetized, spontaneously breathing rats, intravenous bolus injection of 2-APB elicited the pulmonary chemoreflex responses, characterized by apnea, bradycardia, and hypotension. After perineural treatment of both cervical vagi with capsaicin to block the conduction of C fibers, 2-APB no longer evoked any of these reflex responses. In open-chest and artificially ventilated rats, 2-APB evoked an abrupt and intense discharge in vagal pulmonary C fibers in a dose-dependent manner. The stimulation of C fibers by 2-APB was attenuated but not abolished by capsazepine, a selective antagonist of the TRPV1, which completely blocked the response to capsaicin in these C fiber afferents. In isolated pulmonary capsaicin-sensitive neurons, 2-APB concentration dependently evoked an inward current that was partially inhibited by capsazepine but almost completely abolished by ruthenium red, an effective blocker of all TRPV channels. In conclusion, 2-APB evokes a consistent and distinct stimulatory effect on pulmonary C fibers in vivo and on isolated pulmonary capsaicin-sensitive neurons in vitro. These results establish the functional evidence demonstrating that TRPV1, V2, and V3 channels are expressed on these sensory neurons and their terminals.     

Role of TRPV receptors in respiratory diseases

Transient receptor potential vanilloid type channels (TRPVs) are expressed in several cell types in human and animal lungs. Increasing evidence has demonstrated important roles of these cation channels, particularly TRPV1 and TRPV4, in the regulation of airway function. These TRPVs can be activated by a number of endogenous substances (hydrogen ion, certain lipoxygenase products, etc.) and changes in physiological conditions (e.g., temperature, osmolarity, etc.). Activation of these channels can evoke Ca(2+) influx and excitation of the neuron. TRPV1 channels are generally expressed in non-myelinated afferents innervating the airways and lungs, which also contain sensory neuropeptides such as tachykinins. Upon stimulation, these sensory nerves elicit centrally-mediated reflex responses as well as local release of tachykinins, and result in cough, airway irritation, reflex bronchoconstriction and neurogenic inflammation in the airways. Recent studies clearly demonstrated that the excitability of TRPV1 channels is up-regulated by certain autacoids (e.g., prostaglandin E(2), bradykinin) released during airway inflammatory reaction. Under these conditions, the TRPV1 can be activated by a slight increase in airway temperature or tissue acidity. Indirect evidence also suggests that TRPV channels may play a part in the pathogenesis of certain respiratory diseases such as asthma and chronic cough. Therefore, the potential use of TRPV antagonists as a novel therapy for these diseases certainly merits further investigation.     

2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3

The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation.     

Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch

TRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of “forefront” signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance.     

The Thermosensitive Potassium Channel TREK-1 Contributes to Coolness-Evoked Responses of Grueneberg Ganglion Neurons

Neurons of the Grueneberg ganglion (GG) residing in the vestibule of the murine nose are activated by cool ambient temperatures. Activation of thermosensory neurons is usually mediated by thermosensitive ion channels of the transient receptor potential (TRP) family. However, there is no evidence for the expression of thermo-TRPs in the GG, suggesting that GG neurons utilize distinct mechanisms for their responsiveness to cool temperatures. In search for proteins that render GG neurons responsive to coolness, we have investigated whether TREK/TRAAK channels may play a role; in heterologous expression systems, these potassium channels have been previously found to close upon exposure to coolness, leading to a membrane depolarization. The results of the present study indicate that the thermosensitive potassium channel TREK-1 is expressed in those GG neurons that are responsive to cool temperatures. Studies analyzing TREK-deficient mice revealed that coolness-evoked responses of GG neurons were clearly attenuated in these animals compared with wild-type conspecifics. These data suggest that TREK-1 channels significantly contribute to the responsiveness of GG neurons to cool temperatures, further supporting the concept that TREK channels serve as thermoreceptors in sensory cells. Moreover, the present findings provide the first evidence of how thermosensory GG neurons are activated by given temperature stimuli in the absence of thermo-TRPs.     

Identification of a tetrameric assembly domain in the C terminus of heat-activated TRPV1 channels

Transient receptor potential (TRP) channels as cellular sensors are thought to function as tetramers. Yet, the molecular determinants governing channel multimerization remain largely elusive. Here we report the identification of a segment comprising 21 amino acids (residues 752-772 of mouse TRPV1) after the known TRP-like domain in the channel C terminus that functions as a tetrameric assembly domain (TAD). Purified recombinant C-terminal proteins of TRPV1-4, but not the N terminus, mediated the protein-protein interaction in an in vitro pulldown assay. Western blot analysis combined with electrophysiology and calcium imaging demonstrated that TAD exerted a robust dominant-negative effect on wild-type TRPV1. When fused with the membrane-tethered peptide Gap43, the TAD blocked the formation of stable homomultimers. Calcium imaging and current recordings showed that deletion of the TAD in a poreless TRPV1 mutant subunit suppressed its dominant-negative phenotype, confirming the involvement of the TAD in assembly of functional channels. Our findings suggest that the C-terminal TAD in TRPV1 channels functions as a domain that is conserved among TRPV1-4 and mediates a direct subunit-subunit interaction for tetrameric assembly.