Activation properties of heterologously expressed mammalian TRPV2: evidence for species dependence

TRPV2 has been proposed as a potential pain target, in part due to its relatedness to the nociceptor TRPV1 and to its reported activation by noxious high temperatures (>52 degrees C). However, TRPV2 responses to heat as well as to the nonselective agonist 2-aminoethoxydiphenyl borate (2-APB) have not been universally reproduced in other laboratories, leading to debate about the activation properties of this channel. Here, we report the expression of rat, mouse, and human TRPV2 in HEK293 cells and the differential properties of their responses to heat and 2-APB. Expression of mouse or rat TRPV2 in HEK293 cells resulted in robust channel activation when induced by either temperature (>53 degrees C) or 2-APB. By contrast, expression of human TRPV2 did not lead to detectable activation by either of these stimuli. Human TRPV2 protein was expressed at levels comparable with those of rat TRPV2, exhibited similar surface localization and responded to a novelly identified TRPV2 agonist, Delta(9)-tetrahydrocannabinol, indicating that human TRPV2 is functionally expressed on the cell surface. Studies using deletion mutants and chimeras between rat and human TRPV2 indicated that both amino- and carboxyl-cytoplasmic termini of rat TRPV2 are important for responses to heat and 2-APB but can be supplied in trans to form an active channel. The present study not only confirms and extends previous reports demonstrating that rat and mouse TRPV2 respond to 2-APB and noxious heat but also indicates that further investigation will be required to elucidate TRPV2 activation and regulatory mechanisms.     

Electrophysiological properties of heteromeric TRPV4-C1 channels

We previously reported that TRPV4 and TRPC1 can co-assemble to form heteromeric TRPV4-C1 channels [12]. In the present study, we characterized some basic electrophysiological properties of heteromeric TRPV4-C1 channels. 4α-Phorbol 12,13-didecanoate (4α-PDD, a TRPV4 agonist) activated a single channel current in HEK293 cells co-expressing TRPV4 and TRPC1. The activity of the channels was abrogated by a TRPC1-targeting blocking antibody T1E3. Conductance of the channels was ~95pS for outward currents and ~83pS for inward currents. The channels with similar conductance were also recorded in cells expressing TRPV4-C1 concatamers, in which assembled channels were expected to be mostly 2V4:2C1. Fluorescence Resonance Energy Transfer (FRET) experiments confirmed the formation of a protein complex with 2V4:2C1 stoichiometry while suggesting an unlikeliness of 3V4:1C1 or 1V4:3C1 stoichiometry. Monovalent cation permeability profiles were compared between heteromeric TRPV4-C1 and homomeric TRPV4 channels. For heteromeric TRPV4-C1 channels, their permeation profile was found to fit to Eisenman sequence VI, indicative of a strong field strength cation binding site, whereas for homomeric TRPV4 channels, their permeation profile corresponded to Eisenman sequence IV for a weak field strength binding site. Compared to homomeric TRPV4 channels, heteromeric TRPV4-C1 channels were slightly more permeable to Ca2+ and had a reduced sensitivity to extracellular Ca2+ inhibition. In summary, we found that, when TRPV4 and TRPC1 were co-expressed in HEK293 cells, the predominate assembly type was 2V4:2C1. The heteromeric TRPV4-C1 channels display distinct electrophysiological properties different from those of homomeric TRPV4 channels.     

An alternative splicing product of the murine trpv1 gene dominant negatively modulates the activity of TRPV1 channels

Transient receptor potential vanilloid 1 (TRPV1), or vanilloid receptor 1, is the founding member of the vanilloid type of TRP superfamily of nonselective cation channels. TRPV1 is activated by noxious heat, acid, and alkaloid irritants as well as several endogenous ligands and is sensitized by inflammatory factors, thereby serving important functions in detecting noxious stimuli in the sensory system and pathological states in different parts of the body. Whereas numerous studies have been carried out using the rat and human TRPV1 cDNA, the mouse TRPV1 cDNA has not been characterized. Here, we report molecular cloning of two TRPV1 cDNA variants from dorsal root ganglia of C57BL/6 mice. The deduced proteins are designated TRPV1alpha and TRPV1beta and contain 839 and 829 amino acids, respectively. TRPV1beta arises from an alternative intron recognition signal within exon 7 of the trpv1 gene. We found a predominant expression of TRPV1alpha in many tissues and significant expression of TRPV1beta in dorsal root ganglia, skin, stomach, and tongue. When expressed in HEK 293 cells or Xenopus oocytes, TRPV1alpha formed a Ca(2+)-permeable channel activated by ligands known to stimulate TRPV1. TRPV1beta was not functional by itself but its co-expression inhibited the function of TRPV1alpha. Furthermore, although both isoforms were synthesized at a similar rate, less TRPV1beta than TRPV1alpha protein was found in cells and on the cell surface, indicating that the beta isoform is highly unstable. Our data suggest that TRPV1beta is a naturally occurring dominant-negative regulator of the responses of sensory neurons to noxious stimuli.     

Thermosensation and pain

We feel a wide range of temperatures spanning from cold to heat. Within this range, temperatures over about 43 degrees C and below about 15 degrees C evoke not only a thermal sensation, but also a feeling of pain. In mammals, six thermosensitive ion channels have been reported, all of which belong to the TRP (transient receptor potential) superfamily. These include TRPV1 (VR1), TRPV2 (VRL-1), TRPV3, TRPV4, TRPM8 (CMR1), and TRPA1 (ANKTM1). These channels exhibit distinct thermal activation thresholds (>43 degrees C for TRPV1, >52 degrees C for TRPV2, > approximately 34-38 degrees C for TRPV3, > approximately 27-35 degrees C for TRPV4, < approximately 25-28 degrees C for TRPM8 and <17 degrees C for TRPA1), and are expressed in primary sensory neurons as well as other tissues. The involvement of TRPV1 in thermal nociception has been demonstrated by multiple methods, including the analysis of TRPV1-deficient mice. TRPV2, TRPM8, and TRPA1 are also very likely to be involved in thermal nociception, because their activation thresholds are within the noxious range of temperatures.     

Dual expression of mouse and rat VRL-1 in the dorsal root ganglion derived cell line F-11 and biochemical analysis of VRL-1 after heterologous expression.

The vanilloid-like TRP-channel VRL-1 (TRPV2) is a nonselective cation channel expressed by primary sensory neurons and non-neuronal tissues [Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J and Julius, D. (1999) Nature 398, 436-441]. It is one of the six members of the vanilloid-like TRP-channel family which is now termed the TRPV family [Montell, G., Birnbaumer, L., Flockerzi, V., Bindels, R.J., Brutford, E.A., Caterina, M.J., Clapham, D.E., Harteneck, C., Heller, S., Julius, D., Kojima, I., Mori, Y., Penner, R., Prawitt, D., Scharenberg, A.M., Schultz, G., Shimizu, N. and Zhu, M.X. (2002) Mol. Cell 2, 229-231]. As it is a temperature-gated channel, VRL-1 appears to be functionally related to VR1. In contrast to VR1, VRL-1 is activated at a higher temperature threshold and it does not respond to capsaicin or protons. Here we describe the expression of VRL-1 in the rat dorsal root ganglion-derived cell line F-11, a hybridoma of mouse neuroblastoma (N18TG2) and rat dorsal root ganglion cells. We found by RT-PCR that F-11 cells express not only the rat VRL-1, but also its mouse orthologue in a single cell. The F-11 parental cell line N18TG2 also expressed murine VRL-1. Due to its neuronal character, the DRG-derived F-11 cell line provides an experimental system for the study of VRL-1 biochemistry. However, one has to be aware that both the mouse and the rat protein are expressed simultaneously. Furthermore we cloned VRL-1 from rat brain and analyzed its glycosylation and localization in comparison to the endogenously expressed protein in F-11 cells. In contrast to the endogenous VRL-1 the overexpressed protein is glycosylated. Similar to VR1 the glycosylation is N-linked as shown by an deglycosylation assay. Immunofluorescence analysis of the endogenous VRL-1 in F-11 cells gives only weak signals in the cytoplasm whereas the overexpressed rat VRL-1 appears mainly at the plasma membrane.     

TRPV1 at nerve endings regulates growth cone morphology and movement through cytoskeleton reorganization

While the importance of Ca(2+) channel activity in axonal path finding is established, the underlying mechanisms are not clear. Here, we show that transient receptor potential vanilloid receptor 1 (TRPV1), a member of the TRP superfamily of nonspecific ion channels, is physically and functionally present at dynamic neuronal extensions, including growth cones. These nonselective cation channels sense exogenous ligands, such as resenifera toxin, and endogenous ligands, such as N-arachidonoyl-dopamine (NADA), and affect the integrity of microtubule cytoskeleton. Using TRPV1-transiently transfected F11 cells and embryonic dorsal root ganglia explants, we show that activation of TRPV1 results in growth cone retraction, and collapse and formation of varicosities along neurites. These changes were due to TRPV1-activation-mediated disassembly of microtubules and are partly Ca(2+)-independent. Prolonged activation with very low doses (1 nM) of NADA results in shortening of neurites in the majority of isolectin B4-positive dorsal root ganglia neurones. We postulate that TRPV1 activation plays an inhibitory role in sensory neuronal extension and motility by regulating the disassembly of microtubules. This might have a role in the chronification of pain.     

Unique high sensitivity to heat of axolotl TRPV1 revealed by the heterologous expression system

The thermosensation mechanism plays critical roles in various animals living in different thermal environment. We focused on an axolotl, which is a tailed amphibian originally from Lake Xochimilco area in the Vally of Mexico, and examined its behavior response to heat stimulation. Mild heat at 33 °C induced noxious locomotive activity to axolotls, but the noxious response of another tailed amphibian, Iberian ribbed newt, was not observed at 33 °C. To explore the mechanism for the temperature sensitivity of axolotls, we isolated a cDNA of TRPV1. Using the degenerate primer PCR method, we identified the DNA fragment encoding axolotl TRPV1 (axTRPV1), and then cloned a full-length cDNA. We studied the chemical and thermal sensitivities of axTRPV1 by two-electrode voltage clamp method using Xenopus oocyte expression system. Capsaicin, acid, and 2-aminoethoxydiphenylborane apparently activated axTRPV1 channels in a dose-dependent manner. The analysis of thermal sensitivity showed that axTRPV1 was significantly activated by heat but not by cold. The average temperature threshold for heat-activation was 30.95 ± 0.12 °C. This thermal activation threshold of axTRPV1 is unique and significantly low, when compared with the known thresholds of TRPV1s from various animals. Further, this threshold of axTRPV1 is well consistent with the observation of heat-induced behavior of axolotls at 33 °C, demonstrating that axolotl shows noxious response to mild heat mediated through axTRPV1.     

Tunable calcium current through TRPV1 receptor channels

TRPV1 receptors are polymodal cation channels that open in response to diverse stimuli including noxious heat, capsaicin, and protons. Because Ca2+ is vital for TRPV1 signaling, we sought to precisely measure its contribution to TRPV1 responses and discovered that the Ca2+ current was tuned by the mode of activation. Using patch clamp photometry, we found that the fraction of the total current carried by Ca2+ (called the Pf%) was significantly smaller for TRPV1 currents evoked by protons than for those evoked by capsaicin. Using site-directed mutagenesis, we discovered that the smaller Pf% was due to protonation of three acidic amino acids (Asp646, Glu648, and Glu651) that are located in the mouth of the pore. Thus, in keeping with recent reports of time-dependent changes in the ionic permeability of some ligand-gated ion channels, we now show for the first time that the physiologically important Ca2+ current of the TRPV1 receptor is also dynamic and depends on the mode of activation. This current is significantly smaller when the receptor is activated by a change in pH, owing to atomic scale interactions of H+ and Ca2+ with the fixed negative charge of side chains in the pore.     

Coexpression and activation of TRPV1 suppress the activity of the KCNQ2/3 channel

Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselective cation channel expressed predominantly in peripheral nociceptors. By detecting and integrating diverse noxious thermal and chemical stimuli, and as a result of its sensitization by inflammatory mediators, the TRPV1 receptor plays a key role in inflammation-induced pain. Activation of TRPV1 leads to a cascade of pro-nociceptive mechanisms, many of which still remain to be identified. Here, we report a novel effect of TRPV1 on the activity of the potassium channel KCNQ2/3, a negative regulator of neuronal excitability. Using ion influx assays, we revealed that TRPV1 activation can abolish KCNQ2/3 activity, but not vice versa, in human embryonic kidney (HEK)293 cells. Electrophysiological studies showed that coexpression of TRPV1 caused a 7.5-mV depolarizing shift in the voltage dependence of KCNQ2/3 activation compared with control expressing KCNQ2/3 alone. Furthermore, activation of TRPV1 by capsaicin led to a 54% reduction of KCNQ2/3-mediated current amplitude and attenuation of KCNQ2/3 activation. The inhibitory effect of TRPV1 appears to depend on Ca(2+) influx through the activated channel followed by Ca(2+)-sensitive depletion of phosphatidylinositol 4,5-bisphosphate and activation of protein phosphatase calcineurin. We also identified physical interactions between TRPV1 and KCNQ2/3 coexpressed in HEK293 cells and in rat dorsal root ganglia neurons. Mutation studies established that this interaction is mediated predominantly by the membrane-spanning regions of the respective proteins and correlates with the shift of KCNQ2/3 activation. Collectively, these data reveal that TRPV1 activation may deprive neurons from inhibitory control mediated by KCNQ2/3. Such neurons may thus have a lower threshold for activation, which may indirectly facilitate TRPV1 in integrating multiple noxious signals and/or in the establishment or maintenance of chronic pain.     

Mast-cell degranulation induced by physical stimuli involves the activation of transient-receptor-potential channel TRPV2

A characteristic of mast cells is the degranulation in response to various stimuli. Here we have investigated the effects of various physical stimuli in the human mast-cell line HMC-1. We have shown that HMC-1 express the transient receptor potential channels TRPV1, TRPV2 and TRPV4. In the whole-cell patch-clamp configuration, increasing mechanical stress applied to the mast cell by hydrostatic pressure (-30 to -90 cm H(2)O applied via the patch pipette) induced a current that could be inhibited by 10 microM of ruthenium red. This current was also inhibited by 20 microM SKF96365, an inhibitor that is among TRPV channels specific for the TRPV2. A characteristic of TRPV2 is its activation by high noxious temperature; temperatures exceeding 50 °C induced a similar ruthenium-red-sensitive current. As another physical stimulus, we applied laser light of 640 nm. Here we have shown for the first time that the application of light (at 48 mW for 20 min) induced an SKF96365-sensitive current. All three physical stimuli that led to activation of SKF96365-sensitive current also induced pronounced degranulation in the mast cells, which could be blocked by ruthenium red or SKF96365. The results suggest that TRPV2 is activated by the three different types of physical stimuli. Activation of TRPV2 allows Ca(2+) ions to enter the cell, which in turn will induce degranulation. We, therefore, suggest that TRPV2 plays a key role in mast-cell degranulation in response to mechanical, heat and red laser-light stimulation.     

TRP channels and analgesia

Since cloning and characterizing the first nociceptive ion channel Transient Receptor Potential (TRP) Vanilloid 1 (TRPV1), other TRP channels involved in nociception have been cloned and characterized, which include TRP Vanilloid 2 (TRPV2), TRP Vanilloid 3 (TRPV3), TRP Vanilloid 4 (TRPV4), TRP Ankyrin 1 (TRPA1) and TRP Melastatin 8 (TRPM8), more recently TRP Canonical 1, 5, 6 (TRPC1, 5, 6), TRP Melastatin 2 (TRPM2) and TRP Melastatin 3 (TRPM3). These channels are predominantly expressed in C and Aδ nociceptors and transmit noxious thermal, mechanical and chemical sensitivities. TRP channels are modulated by pro-inflammatory mediators, neuropeptides and cytokines. Significant advances have been made targeting these receptors either by antagonists or agonists to treat painful conditions. In this review, we will discuss TRP channels as targets for next generation analgesics and the side effects that may ensue as a result of blocking/activating these receptors, because they are also involved in physiological functions such as release of vasoactive neuropeptides and regulation of vascular tone, maintenance of the body temperature, gastrointestinal motility, urinary bladder control, etc.     

Targeting the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Assembly Domain Attenuates Inflammation-induced Hypersensitivity

The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief.     

Identification of a Protein Kinase C-dependent phosphorylation site involved in sensitization of TRPV4 channel

Transient Receptor Potential (TRP) proteins are non-selective cation channels performing diverse cellular functions. TRPV1 and TRPV4, two calcium-permeable channels of the vanilloid subfamily of TRP proteins, are activated by various physical and chemical stimuli, including noxious heat and mechanical stress, respectively. These channels are also required for exaggerated sensation of painful stimuli, condition referred to as hyperalgesia, which is frequently associated with inflammation. Phosphorylation of TRPV1, involving Protein Kinase C (PKC) and Protein Kinase A (PKA), appears to be the predominant mechanism for channel sensitization and development of heat hyperalgesia. PKC and PKA pathways have also been implicated in the sensitization of TRPV4, but the respective phosphorylation sites remain unknown. Using mass spectrometry, we report now that TRPV4 is phosphorylated on serine 824 by the PKC-activating phorbol 12-myristate 13-acetate. This phosphorylation is prevented by a PKC inhibitor, confirming the involvement of PKC. Ser824, located in the carboxy-terminal cytosolic tail of TRPV4, is also phosphorylated after activation of the PKA pathway by forskolin, albeit less potently. Substitution of Ser824 with aspartic acid, mimicking phosphorylation at this site, increased TRPV4-mediated calcium influx in resting and in stimulated cells, underlining the importance of this residue in TRPV4 regulation. Thus PKC, and possibly PKA, phosphorylate TRPV4 at Ser824 leading to the enhancement of TRPV4 channel function. Our findings suggest an important role of this phosphorylation in TRPV4 sensitization and the development of hyperalgesia.     

An inhibitor of TRPV1 channels isolated from funnel Web spider venom

Capsaicin receptor channels (TRPV1) are nonselective cation channels that integrate multiple noxious stimuli in sensory neurons. In an effort to identify new inhibitors of these channels we screened a venom library for activity against TRPV1 channels and found robust inhibitory activity in venom from Agelenopsis aperta, a north American funnel web spider. Fractionation of the venom using reversed-phase HPLC resulted in the purification of two acylpolyamine toxins, AG489 and AG505, which inhibit TRPV1 channels from the extracellular side of the membrane. The activity of AG489 was characterized further, and the toxin was found to inhibit TRPV1 channels with a K(i) of 0.3 microM at -40 mV. Inhibition of TRPV1 channels by AG489 is strongly voltage-dependent, with relief of inhibition at positive voltages, consistent with the toxin inhibiting the channel through a pore-blocking mechanism. We used scanning mutagenesis throughout the TM5-TM6 linker, a region thought to form the outer pore of TRPV1 channels, to identify pore mutations that alter toxin affinity. Four mutants dramatically decrease toxin affinity and several mutants increase toxin affinity, consistent with the notion that the TM5-TM6 linker forms the outer vestibule of TRPV1 channels and that AG489 is a pore blocker.     

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.     

Potentiation of TRPV3 channel function by unsaturated fatty acids

Transient receptor potential vanilloid (TRPV) channels are polymodal detectors of multiple environmental factors, including temperature, pH, and pressure. Inflammatory mediators enhance TRPV function through multiple signaling pathways. The lipoxygenase and epoxygenase products of arachidonic acid (AA) metabolism have been shown to directly activate TRPV1 and TRPV4, respectively. TRPV3 is a thermosensitive channel with an intermediate temperature threshold of 31-39 degrees C. We have previously shown that TRPV3 is activated by 2-aminoethoxydiphenyl borate (2APB). Here we show that AA and other unsaturated fatty acids directly potentiate 2APB-induced responses of TRPV3 expressed in HEK293 cells, Xenopus oocytes, and mouse keratinocytes. The AA-induced potentiation is observed in intracellular Ca2+ measurement, whole-cell and two-electrode voltage clamp studies, as well as single channel recordings of excised inside-out and outside-out patches. The fatty acid-induced potentiation is not blocked by inhibitors of protein kinase C and thus differs from that induced by the kinase. The potentiation does not require AA metabolism but is rather mimicked by non-metabolizable analogs of AA. These results suggest a novel mechanism regulating the TRPV3 response to inflammation, which differs from TRPV1 and TRPV4, and involves a direct action of free fatty acids on the channel.     

Comparison of currents activated by noxious heat in rat and chicken primary sensory neurons

The vanilloid receptor 1 (VR1) gene is responsible for both capsaicin-, and low threshold (LT) noxious heat-sensitivity in mammalian primary sensory neurons. Although, birds lack capsaicin-sensitivity they express LT noxious heat-sensitivity. Here, we compared LT noxious heat-activated whole-cell currents produced by rat and chicken cultured dorsal root ganglion neurons in order to find out the similarities and differences in the LT noxious heat transduction mechanisms between the two species. No significant differences between rat and chicken neurons were found in the mean cell diameter of the LT noxious heat-sensitive cells (20.4+/-0.8 microm, n=19 and 20.6+/-0.6 microm, n=11, respectively) and the average threshold (45.7+/-0.5 degrees C, n=19 and 46.1+/-0.7 degrees C, n=11, respectively) and peak amplitude (-2.9+/-0.6 nA, n=19 and -2.1+/-0.6 nA, n=11, respectively) of the heat-evoked responses. The current-voltage curves of the responses both in rat and chicken cells reversed at the same range (-19.5+/-3.8 mV, n=4 and -15.5+/-1. 2 mV, n=3, respectively) and showed strong outward rectification at negative membrane potentials. While all LT noxious heat-sensitive rat cells responded to capsaicin, none of the chicken neurons produced detectable response to it. These findings suggest that a VR1 homologue which lacks to sequence for capsaicin-sensitivity is possibly the LT noxious heat transducer in chicken.     

Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells

We have compared activation by heat of TRPV4 channels, heterogeneously expressed in HEK293 cells, and endogenous channels in mouse aorta endothelium (MAEC). Increasing the temperature above 25 degrees C activated currents and increased [Ca(2+)](i) in HEK293 cells transfected with TRPV4 and in MAEC. When compared with activation of TRPV4 currents by the selective ligand 4alphaPDD (alpha-phorbol 12,13-didecanoate), heat-activated currents in both systems showed the typical biophysical properties of currents through TRPV4, including their single channel conductance. Deletion of the three N-terminal ankyrin binding domains of TRPV4 abolished current activation cells by heat in HEK293. In inside-out patches, TRPV4 could not be activated by heat but still responded to the ligand 4alphaPDD. In MAEC, the same channel is activated under identical conditions as in the HEK expression system. Our data indicate that TRPV4 is a functional temperature-sensing channel in native endothelium, that is likely involved in temperature-dependent Ca(2+) signaling. The failure to activate TRPV4 channels by heat in inside-out patches, which responded to 4alphaPDD, may indicate that heat activation depends on the presence of an endogenous ligand, which is missing in inside-out patches.     

The capsaicin receptor TRPV1 is a crucial mediator of the noxious effects of mustard oil

Mustard oil (MO) is a plant-derived irritant that has been extensively used in experimental models to induce pain and inflammation. The noxious effects of MO are currently ascribed to specific activation of the cation channel TRPA1 in nociceptive neurons. In contrast to this view, we show here that the capsaicin receptor TRPV1 has a surprisingly large contribution to aversive and pain responses and visceral irritation induced by MO. Furthermore, we found that this can be explained by previously unknown properties of this compound. First, MO has a bimodal effect on TRPA1, producing current inhibition at millimolar concentrations. Second, it directly and stably activates mouse and human recombinant TRPV1, as well as TRPV1 channels in mouse sensory neurons. Finally, physiological temperatures enhance MO-induced TRPV1 stimulation. Our results refute the dogma that TRPA1 is the sole nocisensor for MO and motivate a revision of the putative roles of these channels in models of MO-induced pain and inflammation. We propose that TRPV1 has a generalized role in the detection of irritant botanical defensive traits and in the coevolution of multiple mammalian and plant species.