Crystal structure of the human TRPV2 channel ankyrin repeat domain

TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.7 A resolution crystal structure of the human TRPV2 ARD. Our crystal structure reveals a six ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contains extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last two repeats. The TRPV2 ARD provides the first structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.     

Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.     

Molecular mechanism underlying modulation of TRPV1 heat activation by polyols

Sensing noxiously high temperatures is crucial for living organisms to avoid heat-induced injury. The TRPV1 channel has long been known as a sensor for noxious heat. However, the mechanism of how this channel is activated by heat remains elusive. Here we found that a series of polyols including sucrose, sorbitol, and hyaluronan significantly elevate the heat activation threshold temperature of TRPV1. The modulatory effects of these polyols were only observed when they were perfused extracellularly. Interestingly, mutation of residues E601 and E649 in the outer pore region of TRPV1 largely abolished the effects of these polyols. We further observed that intraplantar injection of polyols into the hind paws of rats reduced their heat-induced pain response. Our observations not only suggest that the extracellular regions of TRPV1 are critical for the modulation of heat activation by polyols, but also indicate a potential role of polyols in reducing heat-induced pain sensation.     

Structural analyses of the ankyrin repeat domain of TRPV6 and related TRPV ion channels

Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.     

Evolution of vertebrate transient receptor potential vanilloid 3 channels: opposite temperature sensitivity between mammals and western clawed frogs

Transient Receptor Potential (TRP) channels serve as temperature receptors in a wide variety of animals and must have played crucial roles in thermal adaptation. The TRP vanilloid (TRPV) subfamily contains several temperature receptors with different temperature sensitivities. The TRPV3 channel is known to be highly expressed in skin, where it is activated by warm temperatures and serves as a sensor to detect ambient temperatures near the body temperature of homeothermic animals such as mammals. Here we performed comprehensive comparative analyses of the TRPV subfamily in order to understand the evolutionary process; we identified novel TRPV genes and also characterized the evolutionary flexibility of TRPV3 during vertebrate evolution. We cloned the TRPV3 channel from the western clawed frog Xenopus tropicalis to understand the functional evolution of the TRPV3 channel. The amino acid sequences of the N- and C-terminal regions of the TRPV3 channel were highly diversified from those of other terrestrial vertebrate TRPV3 channels, although central portions were well conserved. In a heterologous expression system, several mammalian TRPV3 agonists did not activate the TRPV3 channel of the western clawed frog. Moreover, the frog TRPV3 channel did not respond to heat stimuli, instead it was activated by cold temperatures. Temperature thresholds for activation were about 16 °C, slightly below the lower temperature limit for the western clawed frog. Given that the TRPV3 channel is expressed in skin, its likely role is to detect noxious cold temperatures. Thus, the western clawed frog and mammals acquired opposite temperature sensitivity of the TRPV3 channel in order to detect environmental temperatures suitable for their respective species, indicating that temperature receptors can dynamically change properties to adapt to different thermal environments during evolution.     

Decrypting the Heat Activation Mechanism of TRPV1 Channel by Molecular Dynamics Simulation

As a prototype cellular sensor, the TRPV1 cation channel undergoes a closed-to-open gating transition in response to various physical and chemical stimuli including noxious heat. Despite recent progress, the molecular mechanism of heat activation of TRPV1 gating remains enigmatic. Toward decrypting the structural basis of TRPV1 heat activation, we performed extensive molecular dynamics simulations (with cumulative simulation time of ～11 μs) for the wild-type channel and a constitutively active double mutant at different temperatures (30, 60, and 72°C), starting from a high-resolution closed-channel structure of TRPV1 solved by cryo-electron microscopy. In the wild-type simulations, we observed heat-activated conformational changes (e.g., expansion or contraction) in various key domains of TRPV1 (e.g., the S2-S3 and S4-S5 linkers) to prime the channel for gating. These conformational changes involve a number of dynamic hydrogen-bond interactions that were validated with previous mutational studies. Next, our mutant simulations observed channel opening after a series of conformational changes that propagate from the channel periphery to the channel pore via key intermediate domains (including the S2-S3 and S4-S5 linkers). The gating transition is accompanied by a large increase in the protein-water electrostatic interaction energy, which supports the contribution of desolvation of polar/charged residues to the temperature-sensitive TRPV1 gating. Taken together, our molecular dynamics simulations and analyses offered, to our knowledge, new structural, dynamic, and energetic information to guide future mutagenesis and functional studies of the TRPV1 channels and development of TRPV1-targeting drugs.     

A combined coarse-grained and all-atom simulation of TRPV1 channel gating and heat activation

The transient receptor potential (TRP) channels act as key sensors of various chemical and physical stimuli in eukaryotic cells. Despite years of study, the molecular mechanisms of TRP channel activation remain unclear. To elucidate the structural, dynamic, and energetic basis of gating in TRPV1 (a founding member of the TRPV subfamily), we performed coarse-grained modeling and all-atom molecular dynamics (MD) simulation based on the recently solved high resolution structures of the open and closed form of TRPV1. Our coarse-grained normal mode analysis captures two key modes of collective motions involved in the TRPV1 gating transition, featuring a quaternary twist motion of the transmembrane domains (TMDs) relative to the intracellular domains (ICDs). Our transition pathway modeling predicts a sequence of structural movements that propagate from the ICDs to the TMDs via key interface domains (including the membrane proximal domain and the C-terminal domain), leading to sequential opening of the selectivity filter followed by the lower gate in the channel pore (confirmed by modeling conformational changes induced by the activation of ICDs). The above findings of coarse-grained modeling are robust to perturbation by lipids. Finally, our MD simulation of the ICD identifies key residues that contribute differently to the nonpolar energy of the open and closed state, and these residues are predicted to control the temperature sensitivity of TRPV1 gating. These computational predictions offer new insights to the mechanism for heat activation of TRPV1 gating, and will guide our future electrophysiology and mutagenesis studies.     

Identification and characterization of a Ca2+ -sensitive interaction of the vanilloid receptor TRPV1 with tubulin

The vanilloid receptor TRPV1 plays a well-established functional role in the detection of a range of chemical and thermal noxious stimuli, such as those associated with tissue inflammation and the resulting pain. TRPV1 activation results in membrane depolarization, but may also trigger intracellular Ca2+ -signalling events. In a proteomic screen for proteins associated with the C-terminal sequence of TRPV1, we identified beta-tubulin as a specific TRPV1-interacting protein. We demonstrate that the TRPV1 C-terminal tail is capable of binding tubulin dimers, as well as of binding polymerized microtubules. The interaction is Ca2+ -sensitive, and affects microtubule properties, such as microtubule sensitivity towards low temperatures and nocodazole. Our data thus provide compelling evidence for the interaction of TRPV1 with the cytoskeleton. The Ca2+ -sensitivity of this interaction suggests that the microtubule cytoskeleton at the cell membrane may be a downstream effector of TRPV1 activation.     

Endocannabinoid activation of the TRPV1 ion channel is distinct from activation by capsaicin

Transient receptor potential vanilloid 1 (TRPV1) ion channel serves as the detector for noxious temperature above 42 °C, pungent chemicals like capsaicin, and acidic extracellular pH. This channel has also been shown to function as an ionotropic cannabinoid receptor. Despite the solving of high-resolution three-dimensional structures of TRPV1, how endocannabinoids such as anandamide and N-arachidonoyl dopamine bind to and activate this channel remains largely unknown. Here we employed a combination of patch-clamp recording, site-directed mutagenesis, and molecular docking techniques to investigate how the endocannabinoids structurally bind to and open the TRPV1 ion channel. We found that these endocannabinoid ligands bind to the vanilloid-binding pocket of TRPV1 in the “tail-up, head-down” configuration, similar to capsaicin; however, there is a unique interaction with TRPV1 Y512 residue critical for endocannabinoid activation of TRPV1 channels. These data suggest that a differential structural mechanism is involved in TRPV1 activation by endocannabinoids compared with the classic agonist capsaicin.     

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.     

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.     

Insights into the roles of conserved and divergent residues in the ankyrin repeats of TRPV ion channels

Ion channels are often modulated by intracellular calcium levels. TRPV1, a channel responsible for the burning pain sensation in response to heat, acid or capsaicin, is desensitized at high intracellular calcium concentrations. We recently identified a multiligand-binding site in the N-terminal ankyrin repeat domain (ARD) of TRPV1 that binds ATP and sensitizes the channel. Calcium-calmodulin binds the same site and is necessary for calcium-mediated TRPV1 desensitization. Here, we examine in more detail the conservation of this TRPV1 multiligand-binding site in other species. Furthermore, using sequence analysis, we determine that the unusually twisted shape of the TRPV1-ARD is likely conserved in other TRPV channels, but not in the ARDs of other TRP subfamilies.     

Insights into the Roles of Conserved and Divergent Residues in the Ankyrin Repeats of TRPV Ion Channels

Ion channels are often modulated by intracellular calcium levels. TRPV1, a channel responsible for the burning pain sensation in response to heat, acid or capsaicin, is desensitized at high intracellular calcium concentrations. We recently identified a multiligand-binding site in the N-terminal ankyrin repeat domain (ARD) of TRPV1 that binds ATP and sensitizes the channel. Calcium-calmodulin binds the same site and is necessary for calcium-mediated TRPV1 desensitization. Here, we examine in more detail the conservation of this TRPV1 multiligand-binding site in other species. Furthermore, using sequence analysis, we determine that the unusually twisted shape of the TRPV1-ARD is likely conserved in other TRPV channels, but not in the ARDs of other TRP subfamilies.     

Analysis of Transient Receptor Potential Ankyrin 1 (TRPA1) in Frogs and Lizards Illuminates Both Nociceptive Heat and Chemical Sensitivities and Coexpression with TRP Vanilloid 1 (TRPV1) in Ancestral Vertebrates

Transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (V1) perceive noxious temperatures and chemical stimuli and are involved in pain sensation in mammals. Thus, these two channels provide a model for understanding how different genes with similar biological roles may influence the function of one another during the course of evolution. However, the temperature sensitivity of TRPA1 in ancestral vertebrates and its evolutionary path are unknown as its temperature sensitivities vary among different vertebrate species. To elucidate the functional evolution of TRPA1, TRPA1s of the western clawed (WC) frogs and green anole lizards were characterized. WC frog TRPA1 was activated by heat and noxious chemicals that activate mammalian TRPA1. These stimuli also activated native sensory neurons and elicited nocifensive behaviors in WC frogs. Similar to mammals, TRPA1 was functionally co-expressed with TRPV1, another heat- and chemical-sensitive nociceptive receptor, in native sensory neurons of the WC frog. Green anole TRPA1 was also activated by heat and noxious chemical stimulation. These results suggest that TRPA1 was likely a noxious heat and chemical receptor and co-expressed with TRPV1 in the nociceptive sensory neurons of ancestral vertebrates. Conservation of TRPV1 heat sensitivity throughout vertebrate evolution could have changed functional constraints on TRPA1 and influenced the functional evolution of TRPA1 regarding temperature sensitivity, whereas conserving its noxious chemical sensitivity. In addition, our results also demonstrated that two mammalian TRPA1 inhibitors elicited different effect on the TRPA1s of WC frogs and green anoles, which can be utilized to clarify the structural bases for inhibition of TRPA1.     

A yeast genetic screen reveals a critical role for the pore helix domain in TRP channel gating

TRP cation channels function as cellular sensors in uni- and multicellular eukaryotes. Despite intensive study, the mechanisms of TRP channel activation by chemical or physical stimuli remain poorly understood. To identify amino acid residues crucial for TRP channel gating, we developed an unbiased, high-throughput genetic screen in yeast that uncovered rare, constitutively active mutants of the capsaicin receptor, TRPV1. We show that mutations within the pore helix domain dramatically increase basal channel activity and responsiveness to chemical and thermal stimuli. Mutation of corresponding residues within two related TRPV channels leads to comparable effects on their activation properties. Our data suggest that conformational changes in the outer pore region are critical for determining the balance between open and closed states, providing evidence for a general role for this domain in TRP channel activation.     

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.     

Molecular cloning of an N-terminal splice variant of the capsaicin receptor. Loss of N-terminal domain suggests functional divergence among capsaicin receptor subtypes

Recently a cDNA clone, vanilloid receptor subtype-1 (VR1), was isolated and found to encode an ion channel that is activated by both capsaicin, the pain producing compound in chili peppers, and by noxious thermal stimuli. Subsequently, two related cDNAs have been isolated, a stretch inactivating channel with mechanosensitive properties and a vanilloid receptor-like protein that is responsive to high temperatures (52-53 degrees C). Here, we report the isolation of a vanilloid receptor 5'-splice variant (VR.5'sv) which differs from VR1 by elimination of the majority of the intracellular N-terminal domain and ankyrin repeat elements. Both VR.5'sv and VR1 mRNA were shown to be expressed in tissues reportedly responsive to capsaicin including dorsal root ganglion, brain, and peripheral blood mononuclear cells. Functional expression of VR.5'sv in Xenopus oocytes and mammalian cells showed no sensitivity to capsaicin, the potent vanilloid resiniferatoxin, hydrogen ions (pH 6.2), or noxious thermal stimuli (50 degrees C). Since VR.5'sv is otherwise identical to VR1 throughout its transmembrane spanning domains and C-terminal region, these results support the hypothesis that the N-terminal intracellular domain is essential for the formation of functional receptors activated by vanilloid compounds and noxious thermal stimuli.     

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.     

Toward elucidating the heat activation mechanism of the TRPV1 channel gating by molecular dynamics simulation

As a key cellular sensor, the TRPV1 cation channel undergoes a gating transition from a closed state to an open state in response to various physical and chemical stimuli including noxious heat. Despite years of study, the heat activation mechanism of TRPV1 gating remains enigmatic at the molecular level. Toward elucidating the structural and energetic basis of TRPV1 gating, we have performed molecular dynamics (MD) simulations (with cumulative simulation time of 3 μs), starting from the high‐resolution closed and open structures of TRPV1 solved by cryo‐electron microscopy. In the closed‐state simulations at 30°C, we observed a stably closed channel constricted at the lower gate (near residue I679), while the upper gate (near residues G643 and M644) is dynamic and undergoes flickery opening/closing. In the open‐state simulations at 60°C, we found higher conformational variation consistent with a large entropy increase required for the heat activation, and both the lower and upper gates are dynamic with transient opening/closing. Through ensemble‐based structural analyses of the closed state versus the open state, we revealed pronounced closed‐to‐open conformational changes involving the membrane proximal domain (MPD) linker, the outer pore, and the TRP helix, which are accompanied by breaking/forming of a network of closed/open‐state specific hydrogen bonds. By comparing the closed‐state simulations at 30°C and 60°C, we observed heat‐activated conformational changes in the MPD linker, the outer pore, and the TRP helix that resemble the closed‐to‐open conformational changes, along with partial formation of the open‐state specific hydrogen bonds. Some of the residues involved in the above key hydrogen bonds were validated by previous mutational studies. Taken together, our MD simulations have offered rich structural and dynamic details beyond the static structures of TRPV1, and promising targets for future mutagenesis and functional studies of the TRPV1 channel. Proteins 2016; 84:1938–1949. © 2016 Wiley Periodicals, Inc.     

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.