Thermotaxis is important for animal survival, but the molecular identities of temperature sensors controlling this behavior have not been determined. dTRPA1 is a heat-activated transient receptor potential (TRP) family ion channel member (Viswanath, 2003) that is essential for thermotaxis in Drosophila. dTrpA1 knockdown eliminates avoidance of elevated temperatures along a thermal gradient. dTRPA1 (Flybase name: Anktm1) is expressed in cells without previously ascribed roles in thermosensation: the results implicate dTRPA1-expressing neurons in mediating thermotaxis. The data suggest that thermotaxis relies upon neurons and molecules distinct from those required for high-temperature nociception. It is proposed that dTRPA1 may control thermotaxis by sensing environmental temperature (Rosenzweig, 2005).

Animals exhibit strong behavioral responses to temperature, and many are able to thermotax, undergoing directed migration guided by differences in temperature. Central to thermotaxis are the abilities to sense environmental temperature and to execute the appropriate behavioral response. Animal thermotaxis has been studied most extensively in the nematode C. elegans, where ablation studies have defined the neuronal circuitry involved in thermotactic behavior and molecular genetic studies have identified several molecules required for the development and function of the thermosensory system. However, the molecular identity of the thermal sensors themselves has remained unknown. Larvae and adults of Drosophila also exhibit strong thermotactic behaviors (Sayeed, 1996; Zars, 2001; L. Liu, 2003). No regulators of Drosophila thermotaxis have been identified at the molecular level, and little is known of the neural circuitry that controls thermotaxis, aside from a small group of terminal organ neurons involved in larval cold avoidance (L. Liu, 2003; Rosenzweig, 2005).

Several classes of molecules have been implicated in potentially mediating temperature sensation and they could be involved in thermotaxis. The two-pore-domain K+- channel TREK-1 (Maingret, 2000) and members of the DEG/EnaC-family of Na+ channels (Askwith, 2001) are regulated by temperature in cultured cells, while mice lacking the ATP-gated cation channel P2X3 are defective for electrophysiological responses to moderate warmth (32°C-45°C) (Souslova, 2000). In addition, several members of the Transient Receptor Potential (TRP) family of ion channels have been shown to act as temperature-responsive ion channels in heterologous cells (Jordt, 2003; Patapoutian, 2003), and mice lacking one of these proteins, the heat-activated TRPV1, have been shown to be defective in a withdrawal response to noxious high temperature as well as thermal hyperalgesia upon inflammation (Caterina, 2000; Davis, 2000). While the mouse, C. elegans, and Drosophila genomes all encode two-pore-domain K+ channels, DEG/EnaC proteins, and TRP proteins, it has not been established whether any of these molecules play important roles in thermotaxis (Rosenzweig, 2005).

The temperature-responsive TRPs (TRPV1-V4, TRPM8, and TRPA1) have been dubbed thermoTRPs and include members of three distinct families of TRP channels: TRPV, TRPM, and TRPA (Jordt, 2003; Patapoutian, 2003). The Drosophila genome encodes two TRPV family members, one TRPM, and four TRPAs. Of these proteins, functions have been described for the TRPVs Inactive and Nanchung, which act together in hearing (Kim, 2003; Gong, 2004), and the TRPA Painless, which mediates larval nociceptive responses to high-temperature mechanical stimulation (Tracey, 2003). One Drosophila TRP protein has been shown to function as a temperature-responsive ion channel in heterologous cells (i.e., it is a thermoTRP), dTRPA1 (formerly dANKTM1) (Viswanath, 2003). dTRPA1 has been shown to be the Drosophila ortholog of the single mammalian TRPA protein TRPA1, and the dTRPA1 channel opens in response to warming (Viswanath, 2003). However, the in vivo function of dTRPA1 (and of its mammalian ortholog) in thermosensory behavior has not been explored (Rosenzweig, 2005).

A novel RNAi-based strategy has been developed for studying thermotactic behavior and this approach was used to demonstrate that the warmth-activated ion channel dTRPA1 is essential for thermotaxis. A novel group of dTRPA1-expressing neurons were identified in the CNS that appear important for thermotactic behavior; the proteins and neurons essential for thermotaxis were found to differ from those previously implicated in high-temperature nociceptive behavior. This work identifies a candidate environmental temperature sensor for thermotaxis and provides a cellular and molecular starting point for the dissection of thermoTRP signaling and thermotaxis in Drosophila. In addition, the RNAi-based strategy described here should be applicable to the study of other behaviors in Drosophila (Rosenzweig, 2005).

Drosophila thermotactic behavior was examined by using a thermal preference assay, placing larvae on a gradient of temperatures warmer than their optimal growth temperature (~24°C) and allowing the larvae to migrate from the release zone of 31°C-35°C into a region of even higher temperature or a region of lower temperature. Wild-type late first/early-second instar larvae rapidly migrate down the thermal gradient into the cooler zone. Some larvae explore the warmer zone but rapidly reorient and head down the gradient. Larval thermotactic behavior in this thermal preference assay was quantified with an avoidance index (AI) (L. Liu, 2003). Wild-type larvae achieved AI scores >0.9 within 2 min, demonstrating strong heat avoidance (Rosenzweig, 2005).

A simple RNA interference (RNAi) strategy was developed to survey whether any Drosophila TRPA, TRPV, or TRPM family members might contribute to larval thermotactic behavior. In this approach, embryos were injected with double-stranded RNAs (dsRNAs) corresponding to the genes of interest, and the resulting larvae were analyzed for their ability to thermotax. The injected animals contained a neuronally expressed green fluorescent protein (GFP) transgene, and dsRNA targeting GFP expression was included in all injections, serving as an internal control for a successful injection. Injection of a mixture of dsRNAs corresponding to the four TRPA family members strongly altered thermotaxis. In contrast, injection of dsRNAs corresponding to the TRPVs Inactive and Nanchung or the TRPM CG30078 had no detectable effect on thermotaxis. These latter experiments served as controls demonstrating that the dsRNA injection procedure itself did not affect thermotaxis. Taken together, these data suggest that one or more Drosophila TRPA family members is required for thermotaxis (Rosenzweig, 2005).

Strikingly, RNAi of dTrpA1 alone strongly disrupts avoidance of elevated temperature. Unlike wild-type larvae, similar numbers of dTrpA1(RNAi) larvae migrate into both warmer and cooler zones, yielding AI scores near zero, and many dTrpA1(RNAi) larvae travel deep into the warmer area. Injection of dsRNA against a second, nonoverlapping region of dTrpA1 also generates AI scores near zero. dTrpA1 knockdown disrupts thermotactic behavior throughout larval life, since third instar dTrpA1(RNAi) larvae also exhibit thermotaxis defects. Injection of dsRNAs against the three other TrpAs had no effect on thermotaxis. The TRPA Painless, which mediates responses to high-temperature mechanical stimulation (Tracey, 2003), a thermosensory behavior potentially distinct from thermotaxis, was examined. Neither late-first instar/early-second instar nor third instar painless mutant larvae (pain¹ and pain³) were defective for thermotaxis in this assay. These data demonstrate that dTrpA1 is absolutely required for proper behavior in the thermal preference assay, providing strong genetic evidence for dTrpA1 involvement in thermotaxis. Furthermore, these data also suggest that the TRPA proteins Painless and dTRPA1 have distinct roles in temperature-regulated behavior (Rosenzweig, 2005).

Therefore, the failure of animals lacking dTRPA1 expression to avoid elevated temperatures mirrors the ability of dTRPA1 to function as a heat-activated ion channel in vitro (Viswanath, 2003). Taken together, these data make dTRPA1 an attractive candidate for an environmental temperature sensor controlling thermotaxis. It will be of interest to explore how the temperature-sensing properties of dTRPA1 observed in cultured cells relate to the physiological and behavioral responses of the intact animal. Since closely related dTRPA1 orthologs are present in the Drosophila pseudoobscura, Bombyx morii, and Anopheles gambiae genomes, as well as the C. elegans genome, TRPA1s could regulate thermotaxis in other insects as well as in C. elegans (Rosenzweig, 2005).

Another Drosophila TRPA protein, Painless, also modulates thermosensory behavior (Tracey, 2003). However, dTRPA1 and Painless appear to have distinct thermosensory functions. While painless mutant larvae are strongly defective in responding to noxious high-temperature mechanical stimulation (Tracey, 2003), painless mutants are normal in thermotaxis assays. Further emphasizing the apparent differences between these different thermosensory behaviors, inhibition of the md-Gal4-expressing neurons disrupts responses to noxious high-temperature mechanical stimulation but has no significant effect on thermotaxis, while inhibition of dTrpA1-Gal4 cells has the opposite effect. Thus the migration of larvae away from moderately elevated temperatures (thermotaxis) and the high-temperature nociceptive response appear to rely upon distinct TRPAfamily members and potentially distinct neural circuits (Rosenzweig, 2005).

Determining the neuronal circuitry required for dTrpA1-dependent thermotaxis is an important goal for the future. dTrpA1-Gal4-mediated inhibition and ablation studies are consistent with a role for dTRPA1-expressing CNS neurons in dTrpA1-dependent thermotaxis. CNS thermal sensors have been proposed within the head and thoracic ganglia of adult bees and cockroaches (Heinrich 1980, 1993; Murphy, 1983) and are found in the vertebrate preoptic-anterior hypothalamus (Boulant 2000). Since small insects (Drosophila larvae are <5 mg) have limited heat capacity, internal sensors of elevated temperature could be quite effective. Most importantly, identification of dTRPA1-expressing cells provides an entry point into defining the neuronal circuitry controlling thermotaxis in flies. It will be of interest to determine how dTRPA1-expressing cells participate in the sensation of environmental temperature, either as primary thermosensory cells or as higher-order neurons involved in processing thermosensory input (Rosenzweig, 2005).

dTRPA1 could have functions in addition to thermotaxis. The strong expression of dTRPA1 in the corpus cardiacum is intriguing since this neuroendocrine gland is involved in temperature-dependent developmental phenomena in some insects, including seasonal polyphenism in butterfly wings, where corpus cardiacum ablation prevents establishment of the summer wing pattern. Furthermore, vertebrate TRPA1 has been proposed as a candidate mechanotransduction channel for hearing (Corey, 2004). Whether dTRPA1 responds to mechanical stimulation is unknown, but future analysis of dTRPA1 function in flies could potentially yield insights into mechanosensation as well as thermosensation (Rosenzweig, 2005).

GENE STRUCTURE

Exons - 15

PROTEIN STRUCTURE

Amino Acids - 1274

Structural Domains

dANKTM1 is more similar to mANKTM1 (32% identical, 54% similar) than to Painless (22% identical, 39% similar) and has 13 predicted amino-terminal ankyrins and six transmembrane domains (Viswanath, 2003).

EVOLUTIONARY HOMOLOGS

Characterization of the TRPV1 receptors

For a review of earlier literature on TRPV1 vanilloid receptors see the painless site.

The vanilloid receptor [transient receptor potential (TRP)V1, also known as VR1] is a member of the TRP channel family. These receptors share a significant sequence homology, a similar predicted structure with six transmembrane-spanning domains (S1-S6), a pore-forming region between S5 and S6, and the cytoplasmically oriented C- and N-terminal regions. Although structural/functional studies have identified some of the key amino acids influencing the gating of the TRPV1 ion channel, the possible contributions of terminal regions to vanilloid receptor function remains elusive. In the present study, C-terminal truncations of rat TRPV1 have been constructed to characterize the contribution of the cytoplasmic C-terminal region to TRPV1 function and to delineate the minimum amount of C tail necessary to form a functional channel. The truncation of 31 residues was sufficient to induce changes in functional properties of TRPV1 channel. More pronounced effects of C-terminal truncation were seen in mutants lacking the final 72 aa. These changes were characterized by a decline of capsaicin-, pH-, and heat-sensitivity; progressive reduction of the activation thermal threshold (from 41.5 to 28.6°C); and slowing of the activation rate of heat-evoked membrane currents (Q10 from 25.6 to 4.7). The voltage-induced currents of the truncated mutants exhibit a slower onset, markedly reduced outward rectification, and significantly smaller peak tail current amplitudes. Truncation of the entire TRPV1 C-terminal domain (155 residues) results in a nonfunctional channel. These results indicate that the cytoplasmic COOH-terminal domain strongly influences the TRPV1 channel activity, and that the distal half of this structural domain confers specific thermal sensitivity (Vlachova, 2003).

Temperature affects functions of all ion channels, but few of them can be gated directly. The vanilloid receptor VR1 provides one exception. As a pain receptor, it is activated by heat >42°C in addition to other noxious stimuli, e.g., acids and vanilloids. Although it is understood how ligand- and voltage-gated channels might detect their stimuli, little is known about how heat could be sensed and activate a channel. In this study, the heat-induced single-channel activity of VR1 was characterized, in an attempt to localize the temperature-dependent components involved in the activation of the channel. At <42°C, openings are few and brief. Raising the ambient temperature rapidly increases the frequency of openings. Despite the large temperature coefficient of the apparent activity (Q₁₀ ~27), the unitary current, the open dwell-times, and the intraburst closures are all only weakly temperature dependent (Q₁₀ < 2). Instead, heat has a localized effect on the reduction of long closures between bursts (Q₁₀ ~7) and the elongation of burst durations (Q₁₀ ~32). Both membrane lipids and solution ionic strength affect the temperature threshold of the activation, but neither diminishes the response. The thermodynamic basis of heat activation is discussed, to elucidate what makes a thermal-sensitive channel unique (B. Liu, 2003).

Mammals detect temperature with specialized neurons in the peripheral nervous system. Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. ANKTM1, a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8, has been characterized. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, noxious cold-sensitive sensory neurons have been identified that also respond to capsaicin but not to menthol (Story, 2003).

Wasabi, horseradish and mustard owe their pungency to isothiocyanate compounds. Topical application of mustard oil (allyl isothiocyanate) to the skin activates underlying sensory nerve endings, thereby producing pain, inflammation and robust hypersensitivity to thermal and mechanical stimuli. Despite their widespread use in both the kitchen and the laboratory, the molecular mechanism through which isothiocyanates mediate their effects remains unknown. Mustard oil is shown in this study to depolarize a subpopulation of primary sensory neurons that are also activated by capsaicin, the pungent ingredient in chilli peppers, and by Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana. Both allyl isothiocyanate and THC mediate their excitatory effects by activating ANKTM1, a member of the TRP ion channel family recently implicated in the detection of noxious cold. These findings identify a cellular and molecular target for the pungent action of mustard oils and support an emerging role for TRP channels as ionotropic cannabinoid receptors (Jordt, 2004).

Six members of the mammalian transient receptor potential (TRP) ion channels respond to varied temperature thresholds. The natural compounds capsaicin and menthol activate noxious heat-sensitive TRPV1 and cold-sensitive TRPM8, respectively. The burning and cooling perception of capsaicin and menthol demonstrate that these ion channels mediate thermosensation. In addition to noxious cold, pungent natural compounds present in cinnamon oil, wintergreen oil, clove oil, mustard oil, and ginger all activate TRPA1 (ANKTM1). Bradykinin, an inflammatory peptide acting through its G protein-coupled receptor, also activates TRPA1. Phospholipase C is an important signaling component for TRPA1 activation. Cinnamaldehyde, the most specific TRPA1 activator, excites a subset of sensory neurons highly enriched in cold-sensitive neurons and elicits nociceptive behavior in mice. Collectively, these data demonstrate that TRPA1 activation elicits a painful sensation and provide a potential molecular model for why noxious cold can paradoxically be perceived as burning pain (Bandell, 2004).

Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. TRP (transient receptor potential) ion channels have been investigated as candidates; one, TRPA1 (also known as ANKTM1), meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself (Corey, 2004).

Several mechanisms have been implicated in underlying the perception of cold, most notably the activation of TRPM8 and TRPA1. Tatiometric calcium imaging was used to reveal a population of neurons in the superior cervical ganglion (SCG) of the mouse that respond to cooling but are insensitive to menthol. The expression of the mRNA transcripts encoding the recently identified noxious cold-sensitive channel TRPA1 but not TRPM8 are expressed in the SCG. These data provide evidence for a population of cold-responsive neurons in the SCG whose cold-responsiveness could be mediated by the activation of TRPA1 and suggest that the sympathetic nervous system may play a direct role in mediating sympathetic responses to cold temperatures (Smith, 2004).

date revised: 20 February 2005

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