InteractiveFly: GeneBrief

Transient receptor potential A1: Biological Overview | Regulation | Developmental Biology | Effects of RNAi | Evolutionary Homologs |References

BIOLOGICAL OVERVIEW

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).

Drosophila TRPA1 channel is required to avoid the naturally occurring insect repellent citronellal

Plants produce insect repellents, such as citronellal, which is the main component of citronellal oil. However, the molecular pathways through which insects sense botanical repellents are unknown. This study shows that Drosophila uses two pathways for direct avoidance of citronellal. The olfactory coreceptor OR83b contributes to citronellal repulsion and is essential for citronellal-evoked action potentials. Mutations affecting the Ca2+-permeable cation channel TRPA1 result in a comparable defect in avoiding citronellal vapor. The TRPA1-dependent aversion to citronellal relies on a G protein (Gq)/phospholipase C (PLC) signaling cascade rather than direct detection of citronellal by TRPA1. Loss of TRPA1, Gq, or PLC causes an increase in the frequency of citronellal-evoked action potentials in olfactory receptor neurons. Absence of the Ca2+-activated K+ channel (BK channel) Slowpoke results in a similar impairment in citronellal avoidance and an increase in the frequency of action potentials. These results suggest that TRPA1 is required for activation of a BK channel to modulate citronellal-evoked action potentials and for aversion to citronellal. In contrast to Drosophila TRPA1, Anopheles gambiae TRPA1 is directly and potently activated by citronellal, thereby raising the possibility that mosquito TRPA1 may be a target for developing improved repellents to reduce insect-borne diseases such as malaria (Kwon, 2010).

Two features of the citronellal responses were found to be abnormal in the trpA1¹ basiconic sensilla ab11a neurons. First, there was a higher citronellal-evoked action potential frequency than in wild-type. Second, there was a defect in deactivation in trpA1¹ ab11a neurons. The same two defective phenotypes were observed in ab11 neurons in the dGqα¹ and norpA^P24 mutants, although only the increase in the evoked responses was clearly different when testing significance by analysis of variance. These results support the conclusion that the dGqα¹, norpA^P24, and trpA1¹ mutations affect the citronellal response in an ORN in ab11 (Kwon, 2010).

The finding that there were increases in the frequency of citronellal-evoked action potentials was unexpected and raised a question as to the basis for these defects. TRPA1 is a Ca²⁺-permeable channel, and because reduced activity of Ca²⁺-activated K⁺ channels (BK channels) increases the frequency of action potential firing, it was of interest to see whether loss of TRPA1 caused reduced BK channel activity. If so, then a mutation in the gene (slowpoke, slo) encoding the fly BK channel might phenocopy the trpA1 phenotype. In support of this model, the slo^f05915 mutation caused an increase in the frequency of citronellal-evoked action potentials and impaired citronellal avoidance. Introduction of UAS-slo-RNAi in combination with either the trpA1-GAL4 or the Or83b-GAL4 resulted in a similar defect in citronellal avoidance (Kwon, 2010).

It is proposed that TRPA1 is required for activity of Slo, which in turn modulates citronellal-induced firing of action potentials. Slo might be required in many ORNs and be regulated by additional TRP channels. In support of this proposal, ab12 also responded to citronellal and displayed a higher frequency of action potentials in slo^f05915 but did not function through a Gqα/PLC/TRPA1 pathway. Knockout of a mammalian TRP channel, TRPC1, also disrupts the activity of a Ca²⁺-activated K⁺ channel (K_Ca) in salivary gland cells, and mutations affecting either TRPC1 or K_Ca result in similar defects in salivary gland secretion. Thus, a role for TRP channels in activating Ca²⁺-activated K⁺ channels might be a common but poorly appreciated general phenomenon that is evolutionarily conserved (Kwon, 2010).

The finding that loss of TRPA1 causes an increase rather than a decrease in citronellal-induced action potentials suggests that there might be a TRPA1 independent-pathway required for generating action potentials in response to citronellal. OR83b is a candidate for functioning in such a pathway, because mutation of Or83b interferes with the ability of the synthetic repellent DEET to inhibit the attraction to food odors. This study found that Or83b¹ mutant flies, or Or83b¹ in trans with a deficiency that uncovers the locus, exhibited an impairment in citronellal avoidance similar to that in trpA1 mutant flies. An Or83b¹ defect in the DART assay was not specific to citronellal, because these flies were also impaired in the response to benzaldehyde. Tested were performed to see whether the frequency of citronellal-induced action potentials was altered in Or83b¹ ab11 sensilla. In contrast to the trpA1 mutant phenotype, none of the mutant Or83b¹ ab11 neurons responded to citronellal (Kwon, 2010).

These data indicate that there are dual pathways required for the response to citronellal. OR83b is necessary for producing citronellal-induced action potentials, and a Gq/PLC/TRPA1 pathway appears to function in the modulation of action potential frequency by activating BK channels. It is suggested that an abnormally high frequency of action potentials may lead to rapid depletion of the readily releasable pools of neurotransmitter, thereby muting the citronellal response. Interestingly, a loss-of-function mutation affecting a worm BK channel also results in a behavioral phenotype—increased resistance to ethanol. Although Drosophila TRPA1 functions downstream of a Gq/PLC signaling pathway, citronellal can also directly activate TRPA1, but with low potency. Nevertheless, because Anopheles gambiae TRPA1 is also expressed in the antenna and is activated directly by citronellal with high potency, it is suggested that mosquito TRPA1 represents a new potential target for in vitro screens for volatile activators that might serve as new types of insect repellents (Kwon, 2010).

Drosophila TRPA1 isoforms detect UV light via photochemical production of H₂O₂

The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. This study shows that two specific isoforms of TRPA1 in Drosophila are H₂O₂ sensitive and that they can detect strong UV light via sensing light-induced production of H₂O₂. Ectopic expression of these H₂O₂-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H₂O₂-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H₂O₂-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. Corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H₂O₂-sensitive dTRPA1 isoforms; they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H₂O₂ production but not conventional phototransduction molecules. Thsese results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H₂O₂. It is speculated that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response (a function of CC cells) when they encounter strong UV, an aversive stimulus for young larvae (Guntur, 2015).

This report describes the finding that UV can activate cells via signaling an H₂O₂-dTRPA1 photochemical pathway. Two specific isoforms of dTRPA1 in Drosophila are H₂O₂ sensitive, and ectopically expressing them is sufficient to confer both H₂O₂ and UV light sensitivity to several types of light-insensitive cells. In particular, expressing the H₂O₂-sensitive TRPA1 isoform in a group of proboscis extension-controlling motor neurons was as potent as ChR2 in permitting light to activate the proboscis extension response. The efficacy of the H₂O₂-dTRPA1 pathway in conferring UV sensitivity by was further confirmed by demonstrating that CC cells, a group of cells that express these isoforms endogenously, were sensitive to UV. CC cells' UV response were shown to critically depended on dTRPA1 and H₂O₂ but did not require the conventional phototransduction molecules or Gr28b. This finding is the first to show that specific isoforms of TRPA1 channels can sense UV (and also blue light) via using their H₂O₂ sensitivity, and it raises the intriguing possibility of using these dTRPA1 isoforms as new optogenetics tool (Guntur, 2015).

What is the mechanism that allows specific dTRPA1 isoforms to sense H₂O₂ and consequently UV? A comparison of the protein sequences of different dTRPA1 isoforms showed that the two H₂O₂-sensitive isoforms contain a stretch of 97 aa at the N terminus that is absent in the insensitive one, whereas at least one of the H₂O₂-sensitive isoforms shares the same C terminus as the insensitive one. Thus, it seems the critical residue(s) that confers H₂O₂/UV sensitivity might reside at the N terminus of the H₂O₂-sensitive isoforms. The cysteine residue at the N terminus is of particular interest because H₂O₂ is known to oxidize cysteine, and covalent modification of cysteine residues has been shown to be able to activate TRPA1. Interestingly, although structure-function analysis of mammalian TRPA1 has implicated that H₂O₂ and AITC may modify the same cysteine residues, the current results suggest that this rule may not apply to Drosophila TRPA1, because at least one of the dTRPA1 isoforms, dTRPA1(B)10a, showed robust AITC sensitivity but little, if any, H₂O₂ sensitivity (Guntur, 2015).

One natural question raised by these findings is that why the C4da neurons require Gr28b to sense UV despite the fact that they might also express the H₂O₂-sensitive isoforms: the 'A' promoter for dTRPA1 has been shown to be active in these neurons. One possibility is that the level of dTRPA1 expression in C4da neurons is too low, because the same dTRPA1 antibody detected a clear signal in CC cells but none in C4da neurons. Another possibility is that C4da neurons may express molecules that inhibit dTRPA1 sensitivity to H₂O₂, or molecules that rapidly degrade H₂O₂. Furthermore, it is also conceivable that C4da neurons may not express the H₂O₂-sensitive isoforms of dTRPA1 despite the fact that the promoter for these isoforms appears active in them. Regardless of the exact reasons for C4da neurons' lack of dependency on H₂O₂ for light sensing, it is speculated their reduced H₂O₂ sensitivity causes them to critically depend on Gr28b to activate dTRPA1 in response to light. It is noted that although recent reports have suggested that C4da can sense H₂O₂ at certain developmental stages, this study found that H₂O₂ sensitivity of C4da was significantly lower than that of CC cells (e.g., CC cells responded well to 5 μM H₂O₂, whereas C4da neurons showed no significant responses to 50 μM H₂O₂). Identifying the exact isoforms of dTRPA1 expressed in C4da neurons and determining how they interact with Gr28b are important next steps to address the question of why light-induced H₂O₂ production is not sufficient to confer light responses in C4da neurons (Guntur, 2015).

What is the physiological relevance of CC cells' UV response? Because CC cells are known to express and release the adipokenetic hormone (AKH) that can accelerate heart rate and mobilize sugar into the hemolymph, and that UV is a known aversive stimulus for young Drosophila larvae, it is proposed that CC cells' UV sensitivity may act to promote stress response when young larvae encounter strong UV. It is worth noting that although the sensitivity of CC cells for UV is not high -- the lowest that has been seen with GCaMP6 reporter is ∼80 μW/mm², it may nonetheless be sensitive enough to detect sunlight on earth because one report has suggested that UV of sunlight may reach ~75 μW/mm² in some regions on earth. In addition, although this work primarily focused on CC cells' UV sensitivity, CC cells responded to strong blue light also. Thus, CC cells likely respond to multiple spectrums of sunlight (blue, violet, UV), and their sensitivity to UV reflects only a fraction of their true light sensitivity. Indeed, electrophysiological recording showed that CC cells were capable of responding to 1 mW/mm² white light emitted from a xenon lamp, a light source whose spectrum resembles that of sunlight received on the earth surface. While CC cells are residing in the ring gland as opposed to the body surface, given the transparency of the larval cuticle and the anatomical location of the ring gland (they are located directly above the brain lobes), it is conceivable that light can readily reach and activate these cells. It is also noted that CC cells from at least one other insect species have also been implicated to sense light as EM analysis revealed that they contained rhabdomeres, the bona fide light-sensing organelles. Thus, light sensitivity may be a common feature of these cells, especially among insects that have transparent cuticles (Guntur, 2015).

The mosquito repellent citronellal directly potentiates Drosophila TRPA1, facilitating feeding suppression

Citronellal, a well-known plant-derived mosquito repellent, was previously reported to repel Drosophila melanogaster via olfactory pathways involving but not directly activating Transient Receptor Potential Ankyrin 1 (TRPA1). This study show that citronellal is a direct agonist for Drosophila and human TRPA1s (dTRPA1 and hTRPA1) as well as Anopheles gambiae TRPA1 (agTRPA1). Citronellal-induced activity is isoform-dependent for Drosophila and Anopheles gambiae TRPA1s. The recently identified dTRPA1(A) and agTRPA1(A) isoforms showed citronellal-provoked currents with EC50s of 1.0 +/- 0.2 and 0.1 +/- 0.03 mM, respectively, in Xenopus oocytes, while the sensitivities of TRPA1(B)s were much inferior to those of TRPA1(A)s. Citronellal dramatically enhanced the feeding-inhibitory effect of the TRPA1 agonist N-methylmaleimide (NMM) in Drosophila at an NMM concentration that barely repels flies. Thus, citronellal can promote feeding deterrence of fruit flies through direct action on gustatory dTRPA1, revealing the first isoform-specific function for TRPA1(A) (Du, 2015).

REGULATION

Characterization of Anktm1/dTrpA1 in heterologous systems

Several members of the TRP (for transient receptor potential) family of ionchannels act as physiological temperature sensors in mammals, but it is not known whether the invertebrate TRP sub-families that are found in the fruitfly Drosophila and the roundworm Caenorhabditis elegans can be directly activated by temperature. The Drosophila orthologue of mammalian ANKTM1, a cold-activated ion channel in mammals, responds to a warming rather than a cooling stimulus. The thermosensing function of these channels is therefore evolutionarily conserved, and they show a surprising flexibility in their response to different temperature ranges. In mammals, four TRPVs (members of the vanilloid subfamily of TRP channels) are activated at distinct heat thresholds (33-52°C), whereas TRPM8 (of the melastatin subfamily) and ANKTM1 are activated at cold (17-25°C) temperatures. However, the molecular mechanisms that underlie thermal preference in Drosophila are not well understood (Viswanath, 2003).

To identify potential invertebrate temperature-activated ion channels, the sequences of predicted TRP channels from Drosophila and C. elegans were examined, focusing on orthologues of the mammalian thermosensitive TRPs. Orthologues were operationally defined as reciprocal best BLAST hits on a comparison of the two genomes. By this definition, mammalian TRPM8 and TRPV1-4 channels do not have invertebrate orthologues. Consistent with this, the C. elegans TRPV homologues, which have diverse sensory functions, are not directly activated by temperature. Mammalian ANKTM1 belongs to a branch of TRP channels that includes four Drosophila and two C. elegans predicted proteins. One of these proteins, Painless, is required for response to noxious thermal and mechanical stimuli in Drosophila larvae, although it is unclear whether this channel is a direct sensor (Tracey, 2003). Another of these relatives is a sequence orthologue of mouse ANKTM1 (mANK-TM1; GenBank accession number AY231177): this Drosophila channel has been termed dANKTM1 (Viswanath, 2003).

A full-length, 3.5-kilobase dANKTM1 complementary DNA was amplified from adult Drosophila RNA by using the polymerase chain reaction with reverse transcription; its behaviour as anion channel was analyzed. Cooling temperature steps did not elicit currents at 70 mV from oocytes expressing dANKTM1, but they did elicit strong inward currents in those that expressed mANKTM1 and human ANKTM1. However, transient currents were consistently activated in response to warming in oocytes expressing dANKTM1, with a threshold of 24-29°C. The heat-activated dANKTM1 current was outwardly rectifying and reversed near 30 mV, indicating that dANKTM1 is a relatively non-selective cation channel, as is mANKTM1 (Story, 2003). In calcium-imaging experiments, dANKTM1-transfected Chinese hamster ovary cells also responded to a warming stimulus, with an activation threshold of about 27°C (Viswanath, 2003).

This is the first characterization of an invertebrate temperature-activated ion channel. Given that Drosophila strongly prefers a temperature of 24°C, the activation threshold of dANKTM1 at about 24-29°C suggests that this ion channel might have a physiological role in heat sensing. Although mANKTM1 and dANKTM1 are sequence orthologues, they do not seem to be functional orthologs -- the former senses cooling, whereas the latter senses warming. The two proteins are 54% similar throughout their length, so it is not obvious which domains are crucial for the warm or cold response. But analysis of chimeric mouse and Drosophila ANKTM1 proteins should help in the mapping of temperature-activation domains of these TRP channels (Viswanath, 2003).

A subset of dopamine neurons signals reward for odour memory in Drosophila

Animals approach stimuli that predict a pleasant outcome. After the paired presentation of an odour and a reward, Drosophila can develop a conditioned approach towards that odour. Despite recent advances in understanding the neural circuits for associative memory and appetitive motivation, the cellular mechanisms for reward processing in the fly brain are unknown. This study shows that a group of dopamine neurons in the protocerebral anterior medial (PAM) cluster signals sugar reward by transient activation and inactivation of target neurons in intact behaving flies. These dopamine neurons are selectively required for the reinforcing property of, but not a reflexive response to, the sugar stimulus. In vivo calcium imaging revealed that these neurons are activated by sugar ingestion and the activation is increased on starvation. The output sites of the PAM neurons are mainly localized to the medial lobes of the mushroom bodies (MBs), where appetitive olfactory associative memory is formed. It is therefore proposed that the PAM cluster neurons endow a positive predictive value to the odour in the MBs. Dopamine in insects is known to mediate aversive reinforcement signals. These results highlight the cellular specificity underlying the various roles of dopamine and the importance of spatially segregated local circuits within the MBs (Liu, 2012).

Reward is positive reinforcement and drives the formation of appetitive associative memory. In insects, octopamine was shown to be involved in reward, whereas specific sets of dopamine neurons were identified to mediate aversive reinforcement. Recent studies in Drosophila suggest that dopamine in the MBs is involved in appetitive odour memory, but the specific role of dopamine and the underlying circuit are unclear (Liu, 2012).

To examine whether the activation of dopamine neurons can substitute for a rewarding stimulus in the formation of an appetitive odour memory, the expression of a thermosensitive cation channel dTRPA1 was targeted to different, but overlapping sets of, dopamine neurons by using two GAL4 drivers, TH-GAL4 and DDC-GAL4. Activation of dTRPA1 in DDC-GAL4 flies during the presentation of an odour resulted in a weak appetitive memory, but robust aversive memory in TH-GAL4 flies. The same activation on starvation induced a much greater appetitive memory in DDC-GAL4/UAS-dTrpA1 flies. Activation of dTRPA1 that was not paired with an odour did not induce appetitive memory. Thermo-activation with the driver HL9-GAL4, a variant of DDC-GAL4, induced similar appetitive memory. Furthermore, TH-GAL80 did not significantly suppress induced memory in DDC-GAL4/UAS-dTrpA1 flies, suggesting that the neurons labelled in DDC-GAL4 but not in TH-GAL4 flies are responsible for signalling reward. As in appetitive memory with sugar, a single thermo-activation using DDC-GAL4 induced persistent appetitive memory, which lasted for up to 24 h (Liu, 2012).

To address when starvation is required for the dTRPA1-induced memory performance, examined the effect of changing motivational states was examined before either training or test by a brief feeding. Appetitive memory was induced on thermo-activation despite feeding before training. If applied before the test, feeding fully suppressed the behavioural expression of 12-h memories. These results suggest that starvation is required for the retrieval, but not the acquisition, of appetitive memory induced by thermo-activation (Liu, 2012).

To explore the role of DDC-GAL4-labelled neurons in mediating the sugar reward, the output of these neurons was blocked using Shi^ts1, which inhibits neuronal output at high temperature. Unlike another known type of dopamine neurons that restricts appetitive memory retrieval, blocking the DDC-GAL4-labelled neurons did not release memory expression in fed flies. Instead, the blockade impaired the acquisition, but not the expression, of the sugar-induced memory. Neither memory performance at the permissive temperature nor sugar preference at the restrictive temperature was impaired (Liu, 2012).

Attempts were made to identify the cells responsible for reward processing. DDC-GAL4 heavily labels the PAM cluster neurons, whereas this cluster is sparsely labelled by TH-GAL4. For selective manipulation of the PAM cluster neurons, a collection of GAL4 driver lines was screened, and R58E02-GAL4 was identified. This driver strongly labels the PAM cluster neurons and glial cells in the optic lobes with little expression elsewhere. Arbours of the PAM neurons in the MBs are largely localized to the medial lobes. The enhancer of R58E02-GAL4 is derived from the first intron of the Drosophila dopamine transporter gene. Consistently, the PAM neurons labelled in R58E02-GAL4 as well as in DDC-GAL4 flies are dopamine immunoreactive with no detectable serotonin labelling. Thermo-activation of the PAM neurons with the use of R58E02-GAL4 induced robust appetitive odour memory in starved flies, whereas the activation itself did not cause any obvious reflexive appetitive behaviour (Liu, 2012).

DDC-GAL4 labels many neurons outside the PAM cluster, including those projecting to the s ganglion, where sweet taste neurons terminate. To address the contribution of the non-PAM cells in DDC-GAL4 flies, R58E02-GAL80, a GAL80 line using the same enhancer integrated at the same genomic location as in R58E02-GAL4, was generated. Combination of R58E02-GAL80 with DDC-GAL4 suppressed transgene expression in most PAM neurons in DDC-GAL4 flies. Thermo-activation with DDC-GAL4/R58E02-GAL80 did not induce appetitive memory, demonstrating the importance of PAM neurons in reward signalling (Liu, 2012).

A transient Shi^ts1 block of the PAM neurons by R58E02-GAL4 impaired the acquisition, but not the expression, of sugar-induced memory. Furthermore, blocking the PAM neurons did not impair the reflexive choice of sugar. Consistently, R58E02-GAL80 rescued the memory impairment of DDC-GAL4/UAS-shi^ts1 flies. Thus, the PAM neurons are necessary and sufficient for signalling the sugar reward (Liu, 2012).

Expression of a presynaptic marker using R58E02-GAL4 demonstrated that input and output sites of the PAM neurons are highly segregated, with presynaptic terminals localized predominantly in the MBs. To address whether the signal from the PAM neurons is mediated by dopamine receptors, these neurons were activated in the background of dumb², a mutant for the dDA1 gene (also known as DopR), which encodes a D1-type dopamine receptor. The previously reported role of dDA1 in the Kenyon cells of the MBs for sugar-induced appetitive memory was confirmed. Because it was hoped to use a GAL4 driver to express dDA1 in Kenyon cells simultaneously with dTRPA1 in the PAM neurons, a LexA driver R58E02-LexA::p65 was generated. It recapitulated the expression pattern in R58E02-GAL4 and was able to induce appetitive memory using LexAop2-dTrpA1. Activation of the PAM neurons failed to induce marked appetitive memory in flies lacking dDA1. Driving wild-type dDA1 expression in α/β and γ Kenyon cells by using the driver MB247-GAL4 restored appetitive memory in R58E02-LexA/LexAop2-dTrpA1 flies. These results indicate the importance of dopamine signalling in the MBs for reward processing, but do not exclude a role for other possible co-transmitters released by the PAM neurons (Liu, 2012).

MB-M3 neurons in the PAM cluster have been identified as important for aversive memory formatio. Both MB-M3 and the reward-signalling PAM neurons were labelled in the same brain, and no overlap was found. This highlights the functional heterogeneity of individual cell types in the PAM cluster (Liu, 2012).

Similarly, different populations of dopamine neurons were made that signal appetitive and aversive reinforcement visible by using R58E02-LexA and TH-GAL4, respectively, and the distribution of their projections in the MBs was examined. The terminals of the PAM and protocerebral posterior lateral (PPL)1 clusters are largely non-overlapping in the MBs and together cover the entire lobes despite the simultaneous expression of R58E02-LexA and TH-GAL4 in a few PAM cluster neurons. Thus, axonal compartments of Kenyon cells are targeted by functionally different dopamine neurons (Liu, 2012).

Given the importance of octopamine signalling in reward processing, the PAM cluster neurons were activated in TβH mutants, which lack octopamine. No marked effect of TβH on appetitive memory induced by activation of the PAM neurons was found, indicating that the PAM neurons act in parallel with or downstream of, but not upstream of, octopamine signalling. Consistently, double labelling of the octopamine and PAM cluster dopamine neurons revealed potential direct contacts of these arbours in the spur of the γ lobe and protocerebral regions, where the putative input and output sites of the PAM and octopamine neurons, respectively, are located. This suggests that octopamine may regulate reward processing by directly modulating the activity of the PAM cluster neurons (Liu, 2012).

To test whether the PAM neurons respond to the sugar reward, in vivo calcium imaging was performed in starved flies expressing the fluorescent calcium reporter GCaMP3. A gustatory stimulation protocol was devised with the unrestrained proboscis that enabled confocal imaging of the PAM terminals in the MBs. Sugar ingestion caused stronger calcium responses than water or a bitter caffeine solution. It was found that the calcium response of the PAM neurons on stimulation with sugar was greatly reduced when flies were fed. Flies can sense sweet taste with their tarsi, but stimulating tarsi with sugar barely activated the PAM neurons, suggesting that sweet substances need to be ingested to trigger the reward signal (Liu, 2012).

These data suggest the existence of a reward circuit in which the PAM neurons integrate gustatory reward and other relevant regulatory inputs, and then convey the summed positive value signal to specific subdomains of the MBs. The MB lobes can be anatomically divided into 35 subdomains that are defined by specific combinations of intrinsic and extrinsic neurons. Distinct sets of dopamine neurons may provide functionally independent local circuits within the MBs, potentially allowing appetitive and aversive modulation of the same odour. The PAM neurons may drive positive associative modulation of concomitant olfactory signals of the Kenyon cells. The dual processing of appetitive and aversive stimuli may be a conserved function of dopamine, highlighting the physiological pleiotropy of a neurotransmitter (Liu, 2012).

Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure

Ion channels contribute to the regulation of dorsal closure in Drosophila, a model system for cell sheet morphogenesis. Ca²⁺ was found to be sufficient to cause cell contraction in dorsal closure tissues, as UV-mediated release of caged Ca²⁺ leads to cell contraction. Furthermore, endogenous Ca²⁺ fluxes correlate with cell contraction in the amnioserosa (AS) during closure, whereas the chelation of Ca²⁺ slows closure. Microinjection of high concentrations of the peptide GsMTx4, which is a specific modulator of mechanically gated ion channel function, causes increases in cytoplasmic free Ca²⁺ and actomyosin contractility and, in the long term, blocks closure in a dose-dependent manner. Two channel subunits, ripped pocket and dtrpA1 (TrpA1), were identified that play a role in closure and other morphogenetic events. Blocking channels leads to defects in force generation via failure of actomyosin structures, and impairs the ability of tissues to regulate forces in response to laser microsurgery. These results point to a key role for ion channels in closure, and suggest a mechanism for the coordination of force-producing cell behaviors across the embryo (Hunter, 2014).

These data provide evidence that ion channels function in closure to regulate ion flux in individual cells in the AS and leading edge (LE), leading to Ca2+-dependent cell contractility. Intracellular ion flux via mechanically gated ion channels (MGCs) can promote cytoskeletal and junction organization in cell culture. The findings that localization of the Ca2+ reporter C2:GFP correlates with AS cell contraction and that elevated Ca2+ induces AS cell contraction (via uncaging NP EGTA AM or spontaneous flashes) support a role for Ca2+-dependent contractility in closure. Nevertheless, the observed correlation between perimeter shortening (contraction) and increases in free Ca2+ is by no means perfect. It is hypothesized that there are several reasons for this lack of tight correlation. First, although the data suggest that Ca2+ plays a role in regulating actomyosin contractility, there are other regulators of this important process, and small GTPases (especially Rac and Rho) are sure to play a regulatory role. Ca2+ signaling must be integrated into the context of other signaling pathways and programs of gene expression regulating morphogenesis. Second, individual cell behavior must be considered in the context of the AS cell sheet, in which the behavior of a cell perimeter is profoundly influenced by the behavior of the cells to which it is attached. It is possible that passive perimeter shortening on one side of a given cell is actively driven by contractility in its neighbor. Finally, two-dimensional changes in cell shape, as observed in a given optical section or series of optical sections, must be considered in the context of the three-dimensional nature of cells. It is hypothesized that cell volume does not fluctuate rapidly because of the relative incompressibility of cellular constituents and because the cell does not rapidly lose or gain volume. Thus, cell volume acts as a buffer and changes in crosssectional area (e.g., measured at the level of junctional belts) may be the consequence of contractile activities functioning elsewhere in the cell. Complete understanding of how MGCs and Ca2+-mediated contraction are integrated into cellular homeostasis and morphogenesis requires a more complete picture of how other signaling pathways contribute to changes in cell shape. Moreover, it will require more complete imaging sets, with higher temporal and spatial resolution, of the three-dimensional changes that occur during morphogenesis, even in relatively simple morphogenetic movements such as dorsal closure. The advent of new biosensors and high-speed imaging techniques place the technologies required for such investigations of morphogenesis within the realm of possibility (Hunter, 2014).

GsMTx4 is the most specific pharmacological reagent for manipulating MGC activity in vitro and in vivo, and this study reports its use during Drosophila embryogenesis. Acute, bimodal effects of GsMTx4 on closure are consistent with the presence of MGCs that ultimately pass Ca2+ ions. At the microM concentrations of GsMTx4 experienced by cells at or near the site of injection, increases in cellular free Ca2+ are followed by constriction over the course of tens of seconds. By contrast, the long-term effects of low concentrations of GsMTx4, which cells experience after the bolus of peptide diffuses away from the injection site, appear to be inhibition of closure via the failure of key actomyosin structures and activities. It is hypothesized that GsMTx4 affects MGC activity by modifying the thickness or curvature of the lipid bilayer in which these channels are embedded, consistent with known mechanisms of GsMTx4 action. Studies in cell culture demonstrate that loss of MGC function by pharmacological inhibitors or targeted mutations in channel subunits leads to defects in actomyosin contractile behaviors. Nevertheless, it cannot be ruled out that possible indirect effects of MGC inhibition obscure specific and direct long-term effects of MGC inhibition (e.g. the effect of membrane thickness and curvature on non-MGC membrane proteins during development or secondary consequences of inhibiting RPK and dTRPA1) (Hunter, 2014).

Long-term phenotypes due to GsMTx4-mediated MGC inhibition are recapitulated by RNAi expression or mutational analysis that disrupt the function of specific channels. Congenital loss of channel expression is a long-term effect, and disrupting expression of rpk or dtrpA1 in embryos leads to closure defects. Discrepancies in phenotypes may be the consequence of multiple channels functioning in closure or to differences in the timing and pattern of knockdown or inhibition. Whereas in the current experiments, the expression knockdown of a single channel subunit tissue specifically (via RNAi) or in the embryo as a whole prior to closure (via mutant allele), an advantage of pharmacological inhibitors is acute delivery before or during closure. Indeed, phenotypes were observed consistent with genetic knockdown when GsMTx4 was knocked down prior to closure: defects in AS shape, canthus and purse string formation and failure to close. It is speculated that the embryo can compensate for the congenital loss of a single channel subunit (as in the case of dtrpA1) in ways not possible when drug is applied acutely or when RNAi knocks down channel function (less acutely than the drug, more acutely than inherited mutant alleles). The regulation of contractility via ion channels during closure appears to be both cell-autonomous and non-cell-autonomous (Hunter, 2014).

Specifically, the loss of leading edge cell elongation and their purse strings when channel subunits are targeted in the AS indicates that robust channel activity in the AS is required for normal cell shape changes in both the AS (i.e. cell-autonomous) and in leading edge cells (i.e. non-cell-autonomous). Actomyosin contractility during closure can act non-cell-autonomously, implicating positive reinforcement of force-producing activities or structures between and within embryonic tissues: clones of cells expressing myosin because of a transgenic mosaic effect contract (cell-autonomous effect) but stretch neighboring cells (non-cell-autonomous effect). It is hypothesized that channel activity contributes to tension at a single-cell level in the AS, and that tension in the AS, exerted on the LE, is required for wild-type actomyosin-dependent structures and cell shapes in the leading edge of the LE (Hunter, 2014).

Verification of a mechanical circuit(s) regulated by MGCs requires that which channels are involved can be unequivocally established, and the gating mechanisms of each channel(s) be determined in the embryonic epithelia. The sensitivity of DEG/ENaC and TRPA1 homologs to applied force has been studied in other systems, but is unknown for dorsal closure tissues. Future studies should include electrophysiological recordings, but such methods have not yet been developed for analysis of Drosophila embryonic epithelial cells. These studies could be key for understanding how a Na+-permeable channel (RPK) contributes to Ca2+ flux. Although its ability to conduct Ca2+ or associate with Ca2+ channel subunits is unknown, RPK is involved in Ca2+-dependent processes such as Drosophila oocyte activation and the response to gentle touch in larvae . This study implicates ion flux and MGCs in the molecular mechanisms that regulate closure. Force sensing by MGCs could constitute a rapid means of affecting cell behaviors in order to adapt to acute changes during closure. For example, at the level of apical junctions, individual AS cells change shape dramatically, whereas the overall area of the AS decreases slowly and monotonically. Based on the current observations, it is hypothesized that MGCs function in a mechanical circuit(s) to coordinate forces across the embryo. Similar feedback loops are proposed for the oscillatory behavior of other mechanically coupled, contractile cells. Given that morphogenesis throughout Drosophila development requires the assembly and regulation of force-producing structures, it will be interesting to determine how other morphogenetic processes are affected by channel inhibition (Hunter, 2014).

A switch in thermal preference in Drosophila larvae depends on multiple rhodopsins

Drosophila third-instar larvae exhibit changes in their behavioral responses to gravity and food as they transition from feeding to wandering stages. Using a thermal gradient encompassing the comfortable range (18°C to 28°C), this study found that third-instar larvae exhibit a dramatic shift in thermal preference. Early third-instar larvae prefer 24°C, which switches to increasingly stronger biases for 18°C-19°C in mid- and late-third-instar larvae. Mutations eliminating either of two rhodopsins, Rh5 and Rh6, wipe out these age-dependent changes in thermal preference. In larvae, Rh5 and Rh6 are thought to function exclusively in the light-sensing Bolwig organ. However, the Bolwig organ was found to be dispensable for the thermal preference. Rather, Rh5 and Rh6 are required in trpA1-expressing neurons in the brain, ventral nerve cord, and body wall. Because Rh1 contributes to thermal selection in the comfortable range during the early to mid-third-instar stage, fine thermal discrimination depends on multiple rhodopsins (Sokabe, 2016).

It is concluded that third-instar Drosophila larvae undergo an age-dependent change in their thermal preference, and this behavioral modification requires. Rh5 and Rh6 were the most important, given that the stage-dependent alteration in temperature selection was eliminated in either rh5 and rh6 mutant flies. Several observations support the conclusion that the thermotaxis exhibited by the rh5 and rh6 mutants are not secondary consequences of developmental defects or motor problems. The percentage of larvae that entered the third-instar larval stage at 74 hr AEL was similar to controls, as were the times to pupation. Furthermore, the morphology of the peripheral trpA1-positive neurons that normally express rh5 and rh6 were indistinguishable between the rh5 and rh6 mutants and controls. In addition, the movement speeds of the rh5 and rh6 mutants were not reduced, and they were able to choose 18°C over 28°C normally in two-way choice assays (Sokabe, 2016).

The requirements for Rh5 and Rh6 were light independent, since the thermotaxis occurred equally well in the light or dark and was not dependent on the Bolwig organ, which is the rhodopsin expressing light-sensitive tissue in larvae. Rhodopsins are composed of the protein subunit, opsin and a vitamin-A-derived chromophore, which senses light. In Drosophila photoreceptor cells, the chromophore also functions as a molecular chaperone to facilitate transport of the opsin out of the endoplasmic reticulum. This study found that thermotaxis in late third-instar larvae was impaired in a mutant that disrupts chromophore. However, it is suggested that this phenotype is due to the second function of the chromophore as a molecular chaperone (Sokabe, 2016).

The findings lead to the conclusion that Rh5 and Rh6 function upstream of a Gq/PLC/TRPA1 signaling cascade, which allows late third-instar larvae to select their favorite temperature in the comfortable range. It is proposed that this pathway enables the animals to sense minute temperature differences over a shallow thermal gradient through signal amplification, similar to the role of these proteins in phototransduction. If the perfect option is not available in the thermal landscape, the thermosensory signaling cascade may facilitate adaptation to hospitable temperatures that deviate slightly from their preferred temperature (Sokabe, 2016).

Because of the exquisite effectiveness of rhodopsin in photon capture, it is suggested that Rh5 and Rh6 are expressed outside the Bolwig organ at extremely low levels to prevent light from interfering with temperature sensation. Nevertheless, expression of the rh5 and rh6 reporters was observed in a subset of trpA1-CD neurons in the body wall. Using the GAL4/UAS system, evidence is provided that rh5 and rh6 both function in trpA1-CD- as well as trpA1-AB-expressing neurons outside of the Bolwig organ. In addition, rh5 GAL4-mediated RNAi knockdown of rh6 and rh6 GAL4-mediated knockdown of rh5 resulted in defects in 18°C selection. RNAi-based knockdown of trpA1 with either of the rh5- and rh6-GAL4 drivers caused similar thermotaxis defects. Although these drivers are expressed at very low levels, it is suggested that they are still effective, since trpA1 is also expressed at very low levels in the periphery. The effects of the rh5- and rh6-GAL4 drivers in suppressing trpA1 were not non-specific, as no thermotaxis phenotype was observed using the trp-GAL4 driver. It was also found that the rh5- and rh6-GAL4s silenced the thermosensory neurons in combination with UAS-kir2.1. It is proposed that this was effective, since small increases in hyperpolarization due to slight elevation of Kir2.1 cannot be overcome by the slight depolarization mediated by the low levels of TRPA1 (Sokabe, 2016).

The combination of these findings indicates that both rh5 and rh6 are co-expressed and function in the same, or overlapping, subsets of neurons required for thermotaxis. These findings raise the possibility that Rh5 and Rh6 may form heterodimers in vivo. Another key question is whether rhodopsins are direct thermosensors, an issue that remains unresolved due to challenges inherent in expressing these and most invertebrate rhodopsins in vitro (Sokabe, 2016).

The observation that multiple rhodopsins function in thermotaxis in Drosophila raise the question as to whether rhodopsin-dependent thermosensory signaling cascades are used in other animals, including mammals. It is suggested that mammalian cells that undergo thermotaxis over very small temperature gradients may rely on opsin-coupled amplification cascades. Intriguing possibilities include leukocytes, which thermotax to sites of inflammation, and mammalian sperm, which undergo thermotaxis to the egg over temperature gradients of ~1°C and require PLC for this cellular behavior. Intriguingly, mammalian TRP channels and non-visual rhodopsins appear to be expressed in sperm and have been suggested to function in sperm thermotaxis (Sokabe, 2016).

Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut

Precise regulation of stem cell activity is crucial for tissue homeostasis and necessary to prevent overproliferation. In the Drosophila adult gut, high levels of reactive oxygen species (ROS) has been detected with different types of tissue damage, and oxidative stress has been shown to be both necessary and sufficient to trigger intestinal stem cell (ISC) proliferation. However, the connection between oxidative stress and mitogenic signals remains obscure. In a screen for genes required for ISC proliferation in response to oxidative stress, this study identified two regulators of cytosolic Ca2+ levels, transient receptor potential A1 (TRPA1) and ryanodine receptor (RyR). Characterization of TRPA1 and RyR demonstrates that Ca2+ signaling is required for oxidative stress-induced activation of the Ras/MAPK pathway, which in turns drives ISC proliferation. These findings provide a link between redox regulation and Ca2+ signaling and reveal a novel mechanism by which ISCs detect stress signals (Xu, 2017).

This study found that the two cation channels TRPA1 and RyR are critical for cytosolic Ca2+ signaling and ISC proliferation. Under homeostatic conditions, the basal activities of TRPA1 and RyR are required for maintaining cytosolic Ca2+ in ISCs to ensure their self-renewal activities and normal tissue turnover. Agonists, including but not limited to low levels of ROS, could be responsible for the basal activities of TRPA1 and RyR. Under tissue damage conditions, increased ROS stimulates the channel activities of TRPA1 to mediate increases in cytosolic Ca2+ in ISCs. As for RyR, besides its potential to directly sense ROS, it is known to act synergistically with TRPA1 in a positive feedback mechanism to release more Ca2+ from the ER into the cytosol upon sensing the initial Ca2+ influx through TRPA1 (Xu, 2017).

Previously, Deng (2015) identified L-glutamate as a signal that can activate metabotropic glutamate receptor (mGluR) in ISCs, which in turn modulates the cytosolic Ca2+ oscillation pattern via phospholipase C (PLC) and inositol-1,4,5-trisphosphate (InsP3). Interestingly, L-glutamate and mGluR RNAi mainly affected the frequency of Ca2+ oscillation in ISCs, while their influence on cytosolic Ca2+ concentration was very weak. Strikingly, the number of mitotic cells induced by L-glutamate (i.e. an increase from a basal level of ~5 per midgut to ~10 per midgut) is far less than what has been observed in tissue damage conditions (depending on the severity of damage, the number varies from ~20 to more than 100 per midgut following damage). Consistent with this, in a screen for regulators of ISC proliferation in response to tissue damage, this study tested three RNAi lines targeting mGluR (BL25938, BL32872, and BL41668, which was used by Deng, 2015), and none blocked the damage response in ISCs, suggesting that L-glutamate and mGluR do not play a major role in damage repair of the gut epithelium (Xu, 2017).

This study found that ROS can trigger Ca2+ increases through the redox- sensitive cation channels TRPA1 and RyR under damage conditions. In particular, it was demonstrated using voltage-clamp experiments that the TRPA1-D isoform, which is expressed in the midgut, is sensitive to the oxidant agent paraquat. In addition, the results of previous studies have demonstrated the direct response of RyR to oxidants via single channel recording and showed that RyR could amplify TRPA1-mediated Ca2+ signaling through the Ca2+-induced Ca2+ release (CICR) mechanism. Interestingly, expression of oxidant- insensitive TRPA1-C isoform in the ISCs also exhibits a tendency to induce ISC proliferation, indicating that ROS may not be the only stimuli for TRPA1 and RyR under physiological conditions. Possible other activators in the midgut may be irritant chemicals, noxious thermal/mechanical stimuli, or G-protein-coupled receptors (Xu, 2017).

Altogether, the concentration of cytosolic Ca2+ in ISCs appears to be regulated by a number of mechanisms/inputs including mGluR and the ion channels TRPA1 and RyR. Although mGluR might make a moderate contribution to cytosolic Ca2+ in ISCs, TRPA1 and RyR have a much stronger influence on ISC Ca2+ levels. Thus, it appears that the extent to which different inputs affect cytosolic Ca2+ concentration correlates with the extent of ISC proliferation (Xu, 2017).

Although, as a universal intracellular signal, cytosolic Ca2+ controls a plethora of cellular processes, we were able to demonstrate that cytosolic Ca2+ levels regulate Ras/MAPK activity in ISCs. Specifically, we found that trpA1 RNAi or RyR RNAi block Ras/MAPK activation in stem cells, and that forced cytosolic Ca2+ influx by SERCA RNAi induces Ras/MAPK activity. Moreover, Ras/MAPK activation is an early event following increases in cytosolic Ca2+, since increased dpErk signal was observed in stem cells expressing SERCA RNAi before they undergo massive expansion, and when Yki RNAi was co-expressed to block proliferation. It should be noted that a more variable pattern of pErk activation was observed with prolonged increases of cytosolic Ca2+, suggesting complicated regulations via negative feedback, cross-activation, and cell communication at late stages of Ca2+ signaling. This might explain why Deng failed to detect pErk activation after 4 days induction of Ca2+ signaling (Deng, 2015). Previously, Ras/MAPK activity was reported to increase in ISCs, regulating proliferation rather than differentiation, in regenerating midguts, which is consistent with the findings about TRPA1 and RyR (Xu, 2017).

The Calcineurin A1/CREB-regulated transcription coactivator/CrebB pathway previously proposed to act downstream of mGluR-calcium signaling (Deng, 2015) is not likely to play a major role in high Ca2+-induced ISC proliferation, as multiple RNAi lines targeting CanA1 or CrebB were tested and none of them suppressed SERCA RNAi-induced ISC proliferation. In support of this model, it was also found that the active forms of CanA1/ CRTC/ CrebB cannot stimulate mitosis in ISCs when their cytosolic Ca2+ levels are restricted by trpA1 RNAi, whereas mitosis induced by the active forms of Ras or Raf is not suppressed by trpA1 RNAi (Xu, 2017).

Prior to this study, it has been shown that paracrine ligands such as Vn from the visceral muscle, and autocrine ligands such as Spi and Pvf ligands from the stem cells, can stimulate ISC proliferation via RTK-Ras/MAPK signaling. It study found that multiple RTK ligands in the midgut are down-regulated by trpA1 RNAi expression in the ISCs, including spi and pvf1 that can be induced by SERCA RNAi. Further, it was demonstrated that high Ca2+ fails to induce ISC proliferation in the absence of EGFR. As spi is induced by EGFR-Ras/MAPK signaling in Drosophila cells, and DNA binding mapping (DamID) analyses indicate that spi might be a direct target of transcriptional factors downstream of EGFR-Ras/MAPK in the ISCs, the autocrine ligand Spi might therefore act as a positive feedback mechanism for EGFR-Ras/MAPK signaling in ISCs (Xu, 2017).

In summary, this study identifies a mechanism by which ISCs sense microenvironment stress signals. The cation channels TRPA1 and RyR detect oxidative stress associated with tissue damage and mediate increases in cytosolic Ca2+ in ISCs to amplify and activate EGFR-Ras/MAPK signaling. In vertebrates, a number of cation channels, including TRPA1 and RyR, have been associated with tumor malignancy. The current findings, unraveling the relationship between redox-sensing, cytosolic Ca2+, and pro-mitosis Ras/MAPK activity in ISCs, could potentially help understand the roles of cation channels in stem cells and cancers, and inspire novel pharmacological interventions to improve stem cell activity for regeneration purposes and suppress tumorigenic growth of stem cells (Xu, 2017).

DEVELOPMENTAL BIOLOGY

Antisera raised against dTRPA1 detected strong dTRPA1 protein expression in a small number of central brain neurons and in neuroendocrine cells of the corpus cardiacum. dTrpA1(RNAi) larvae had no detectable dTRPA1 expression in these regions, demonstrating that the antisera was specific for dTRPA1 and that the dTrpA1 dsRNA effectively reduced dTRPA1 protein expression. Specific dTRPA1 expression was also detected in two pairs of cells adjacent to the mouthhooks and in the developing gut. Interestingly, dTRPA1 expression was not detected in either multiple-dendritic neurons (implicated in temperature- dependent nociceptive responses) (Tracey, 2003) or chordotonal neurons, both of which show temperature- dependent calcium changes (L. Liu, 2003). The role of multiple-dendritic neurons was explored using md-Gal4:UAS-TeTxLC larvae. Consistent with no significant role of multiple-dendritic neurons in the thermotaxis assay, significant thermotaxis defects were not detected in md-Gal4:UAS-TeTxLC larvae. In addition, significant thermotaxis defects were not detected in atonal(RNAi) larvae, which lack chordotonal neurons. These data suggest that there may be differences in the neuronal circuitry required for thermotaxis in response to elevated temperature and withdrawal from a high-temperature nociceptive stimulus (Rosenzweig, 2005).

The role of dTRPA1-expressing cells in thermotaxis was further examined by expressing TeTxLC or the cell death-promoting gene Hid under control of putative dTrpA1 promoter sequences. dTrpA1-Gal4 drives GFP expression in most dTRPA1-expressing central brain neurons, but not in the other dTRPA1-expressing cell populations. dTrpA1-Gal4:UAS-Hid animals had reduced numbers of dTRPA1-expressing brain neurons (e.g., only one of the three dTPRA1-expressing cells was present), while the corpus caridiaca appeared unaffected. Consistent with the participation of dTrpA1-Gal4-expressing cells in thermosensory behavior, dTrpA1-Gal4:UAS-Hid as well as dTrpA1-Gal4:UAS-TeTxLC third instar larvae were partially, but significantly (p < 0.005), compromised in thermotactic behavior (Rosenzweig, 2005).

Interestingly, both dTrpA1-Gal4:UAS-TeTxLC and dTrpA1-Gal4:UAS-Hid larvae exhibit normal withdrawal from a high-temperature nociceptive stimulus, indicating that these animals are not defective for all thermosensory responses. Similar effects were also obtained using a second, independent dTrpA1-Gal4 insertion. These data further support the notion that there may be differences in the neuronal circuitry required for thermotaxis and high-temperature nociception (Rosenzweig, 2005).

TRPA1 channels in Drosophila and honey bee ectoparasitic mites share heat sensitivity and temperature-related physiological functions

TRPA1 is conserved between many arthropods, and in some has been shown to function as a chemosensor for noxious compounds. Activation of arthropod TRPA1 channels by temperature fluctuations has been tested in only a few insect species, and all of them were shown to be activated by heat. The recent identification of chemosensitive TRPA1 channels from two honey bee ectoparasitic mite species (VdTRPA1 and TmTRPA1) have provided an opportunity to study the temperature-dependent activation and the temperature-associated physiological functions of TRPA1 channels in non-insect arthropods. This study found that both mite TRPA1 channels are heat sensitive and capable of rescuing the temperature-related behavioral defects of a Drosophila melanogaster trpA1 mutant. These results suggest that heat-sensitivity of TRPA1 could be conserved between many arthropods despite its amino acid sequence diversity. Nevertheless, the ankyrin repeats (ARs) 6 and 7 are well-conserved between six heat-sensitive arthropod TRPA1 channels and have critical roles for the heat activation of VdTRPA1 (Peng, 2016).

Effects of Mutation, RNAi and Ecotopic Expression

Tests were performed to see how specific the behavioral defect of dTrpA1(RNAi) animals was for thermotaxis. (1) It was determined that the motility indices (MIs) of wild-type and dTrpA1(RNAi) larvae in the thermal gradient were indistinguishable. (The MI represents the fraction of larvae no longer in the release zone at a given time point.) Late-first instar/early-second instar wild-type larvae had MIs of 0.56 and 0.64 at 2 and 5 min after release, while dTrpA1(RNAi) larvae had MIs of 0.60 and 0.69 at these time points. Thus dTrpA1(RNAi) animals are specifically defective in thermotaxis rather than in their ability to migrate at elevated temperature. (2) Larval chemotaxis in response to an olfactory repellent (n-octyl acetate) was examined; dTrpA1(RNAi) larvae were found to respond similarly to wild type, indicating that dTrpA1(RNAi) larvae are not defective for all avoidance behavior. Finally, responsiveness of dTrpA1(RNAi) larvae to higher temperatures (55°C) was examined. Because larvae rapidly die when exposed to gradients of higher temperature, larvae were tested by using a temperature-dependent nociceptive assay (Tracey, 2003). Crawling third instar larvae rapidly and dramatically curl when touched with a hot (55°C) probe but not when touched with a 25°C or 36°C probe. dTrpA1(RNAi) larvae respond indistinguishably from wild-type larvae in this assay (Rosenzweig, 2005).

As a negative control for the assay, larvae expressing tetanus toxin light chain (TeTxLC), an inhibitor of synaptic vesicle release, were examined under md-Gal4 control (md-Gal4 is expressed in multiple-dendritic neurons and ~100 CNS neurons) (Tracey, 2003). As previously reported (Tracey, 2003), md-Gal4:UAS-TeTxLC larvae were defective in response to contact with the hot probe. The ability of dTrpA1(RNAi) larvae to chemotax and respond to other thermal stimuli suggests a specific requirement for dTrpA1 in thermotaxis (Rosenzweig, 2005).

Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. This study identified small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival (Hamada, 2008).

Although the physiology of all cells is affected by temperature, the expression of temperature-activated members of the transient receptor potential (TRP) family (thermoTRPs) can make cell excitability highly temperature-responsive (Dhaka, 2006). ThermoTRPs are cation channels with highly temperature-dependent conductances that participate in thermosensation from insects to humans. The Drosophila TRP channel dTrpA1 promotes larval heat avoidance (Rosenzweig, 2005) and can be activated by warming in ooctyes (Viswanath, 2003). This study addressed whether dTrpA1 contributes to the selection of a preferred temperature in the adult fly. When allowed to distribute along a thermal gradient for 30 min, wild-type D. melanogaster adults prefer ~25°C, their optimal growth temperature. Compared to wild-type controls, dTrpA1 loss-of-function mutant animals showed increased accumulation in the warmest (28-32°C) regions of the gradient, but not in the coolest (18-22°C) regions. A dTrpA1 genomic minigene rescued the phenotype. Animals heterozygous for dTrpA1 loss-of-function mutations also preferred slightly elevated temperatures. Thus, dTrpA1 function is important for determining thermal preference and specifically contributes to avoidance of warm regions (Hamada, 2008).

If dTrpA1 was involved in thermotransduction, it should regulate the warmth responsiveness of thermosensors. As the identity of the adult Drosophila thermosensors was unknown, dTrpA1 protein expression was examined (using anti-dTrpA1 antisera). dTrpA1 expression was detected in three sets of previously uncharacterized cells in the brain: lateral cell (LC), ventral cell (VC) and AC neurons. dTrpA1 was also detected in the proboscis, but ablation studies detected no contribution of the proboscis to warmth avoidance. To focus on the neurons that contribute to thermal preference, where the rescuing dTrpA1 minigene restored dTrpA1 expression was examined; dTrpA1 expression was restored specifically within AC neurons, but not LC or VC neurons. This suggested that dTrpA1 expression in AC neurons (two pairs of neurons at the brain's anterior) sufficed to restore thermal preference and that AC neurons might act as thermosensors (Hamada, 2008).

Temperature responsiveness of AC neurons was examined using the fluorescent calcium indicator G-CaMP. When exposed to increasing temperature, AC neurons showed robust increases in G-CaMP fluorescence, reflecting warmth-responsive increases in intracellular calcium. Ten out of the 27 AC neurons imaged had fluorescence increases between 4% and 39%, with a mean increase over baseline among these cells of 15%. The average temperature at which fluorescence increases were initially observed was 24.9°C, compatible with AC activation as temperatures rise above preferred. In contrast, none of the 21 dTrpA1 mutant AC neurons imaged had fluorescence increases. As a control that mutant AC neurons remained physiologically active, it was confirmed that they showed robust responses on potassium chloride addition. Notably, AC responses did not depend on an intact periphery, since all G-CaMP studies were performed using isolated brains from which peripheral tissues had been removed. These observations identify AC neurons as warmth-activated, dTrpA1-dependent thermosensors (Hamada, 2008).

AC neurons project towards several brain regions, including the antennal lobe. The antennal lobe is implicated in cockroach thermosensation, but has been studied exclusively for olfaction in Drosophila. So far, 11 of the ~50 antennal lobe glomeruli remain unassociated with identified olfactory receptors. AC neurites elaborated within two such unassociated glomeruli, VL2a and VL2p. Thus the Drosophila antennal lobe contains both thermosensory and olfactory neuron processes. VL2a is also innervated by Fruitless-expressing neurons implicated in pheromone transduction, suggesting that even individual glomeruli receive multi-modal sensory information. AC processes also branched within the s ganglion and superior lateral protocerebrum, although these target regions are less defined than in the antennal lobe. These regions have been previously implicated in processing other types of sensory input (Hamada, 2008).

As dTrpA1 expression in AC neurons seemed sufficient to restore normal thermal preference, whether such expression was also necessary was also examined. dTrpA1 was knocked down selectively in AC neurons using tissue-specific RNA interference targeting dTrpA1 controlled by dTrpA1^SH-GAL4, a promoter expressed in AC but not LC or VC neurons. Consistent with the importance of dTrpA1 expression in AC neurons in thermal preference, AC knockdown increased the fraction of animals present in the 28-32°C region compared to controls. Similar results were obtained when dTrpA1 expression was knocked down using a broad neuronal promoter (Appl-GAL4). All knockdowns were assessed by dTrpA1 immunohistochemistry. dTrpA1 knockdown with the general cholinergic neuron promoter Cha(7.4)-GAL4 eliminated detectable dTrpA1 expression in AC (and LC and VC ) neurons, decreasing warmth avoidance. In contrast, dTrpA1 RNAi expressed using Cha(1.2)-GAL4 -- which is expressed in many brain cholinergic neurons but not AC neurons -- did not disrupt warmth avoidance. Taken together, these data suggest that dTrpA1 expression in AC (but not LC or VC) neurons is both necessary and sufficient for normal thermal preference behaviour. Whether LC and VC neurons participate in other warmth-activated responses is unknown (Hamada, 2008).

The identification of an internal sensor controlling temperature preference conflicts with the established view that Drosophila sense moderate warming using thermosensors in the third antennal segment. The effects were tested of surgically removing either one third antennal segment and arista (unilateral ablation) or both (bilateral ablation). Both unilateral and bilateral ablation increased the fraction of animals in cool (18-22°C), but not warm (28-32°C), regions. Thus these tissues were dispensable for warmth avoidance, but essential for cool avoidance. When dTrpA1 mutants were subjected to bilateral ablation, they accumulated in both cool and warm regions: the fraction between 18-22°C did not differ from wild-type ablation animals; the fraction between 28-32°C did not differ from non-ablated dTrpA1 mutants. Thus dTrpA1-expressing cells and antennal cells function additively to set preferred temperature, promoting avoidance of elevated and reduced temperatures, respectively (Hamada, 2008).

These data are consistent with warmth activation of dTrpA1 serving as the molecular basis of warmth sensing by AC neurons. As thermal activation of mammalian TRPA1 proteins is controversial, whether dTrpA1 could act as a molecular sensor of warming in the fly was tested. Indeed, misexpression of dTrpA1 throughout the fly nervous system (using C155-GAL4) caused a dramatic phenotype not observed in controls: heating these flies to 35°C for 60 s caused incapacitation, an effect reversed on return to 23°C. Similar effects were observed using electrophysiology, with moderate warming (above ~25°C) triggering a barrage of excitatory junction potentials at the neuromuscular junction. These data strongly support dTrpA1 acting as a molecular sensor of warming. The ability of dTrpA1 mis-expression to confer warmth activation also suggests that dTrpA1 can be used as a genetically encoded tool for cell-specific, inducible neuronal activation. dTrpA1 might be particularly useful in tissues such as the fly brain where thermal stimulation is easier to deliver than the chemical or optical stimulation that controls other tools for modulating neuronal activity (Hamada, 2008).

To test whether warmth activation is a property of other insect TrpA1s, the malaria mosquito Anopheles gambiae TrpA1 (agTrpA1) was examined. dTrpA1 is warmth-activated when expressed in Xenopus laevis oocytes. agTrpA1 also showed robust warmth activation. These currents were specific, they were not observed in uninjected oocytes and were inhibited by ruthenium red (which antagonizes other TRPs). Similar to mammalian thermoTRPs, both dTrpA1 and agTrpA1 showed outward rectification. Closely related TrpA1s are present in the flour beetle Tribolium castaneum and in disease vectors such as Pediculus humanus corporis (body lice), Culex pipiens (common house mosquito) and Aedes aegypti (yellow and dengue fever mosquito) which use warmth-sensing for host location and habitat selection. Such insect TrpA1s constitute potential targets for disrupting thermal preference and other thermosensory behaviours in agricultural pests and disease vectors (Hamada, 2008).

Environmental temperature affects the physiology of all animals. Increasing temperatures associated with climate change are linked to poleward redistributions of hundreds of species including insects, fish, birds and mammals, AC neurons are internal. As a ~1 mg fly is readily penetrated by ambient temperature variations, such an internal sensor should monitor environmental temperature effectively. dTrpA1 activation seems to be critical for AC neuron activation, suggesting that dTrpA1 threshold and expression changes could modulate thermal preferences. More speculatively, changes in insect TrpA1 function and expression could facilitate movements into novel environments or development of novel behaviours such as host seeking (Hamada, 2008).

Although effects of environmental temperature on behaviour are ubiquitous, the mechanisms animals use to seek out optimal temperatures are largely unknown. AC neurons become active as temperatures rise above the preferred temperature, suggesting that they may function as 'discomfort' receptors that, together with putative antennal cool receptors (similar to those described in other insect antennae), repel the fly from all but the most optimal temperatures. Notably, mice lacking the cool-activated channel TRPM8 prefer abnormally cool temperatures, whereas mice lacking heat-activated TRPV4 prefer warmer temperatures, indicating that similar strategies may be used in mammals (Hamada, 2008).

Drosophila TRPA1 channel is required to avoid the naturally occurring insect repellent citronellal

Plants produce insect repellents, such as citronellal, which is the main component of citronellal oil. However, the molecular pathways through which insects sense botanical repellents are unknown. This study shows that Drosophila uses two pathways for direct avoidance of citronellal. The olfactory coreceptor OR83b contributes to citronellal repulsion and is essential for citronellal-evoked action potentials. Mutations affecting the Ca2+-permeable cation channel TRPA1 result in a comparable defect in avoiding citronellal vapor. The TRPA1-dependent aversion to citronellal relies on a G protein (Gq)/phospholipase C (PLC) signaling cascade rather than direct detection of citronellal by TRPA1. Loss of TRPA1, Gq, or PLC causes an increase in the frequency of citronellal-evoked action potentials in olfactory receptor neurons. Absence of the Ca2+-activated K+ channel (BK channel) Slowpoke results in a similar impairment in citronellal avoidance and an increase in the frequency of action potentials. These results suggest that TRPA1 is required for activation of a BK channel to modulate citronellal-evoked action potentials and for aversion to citronellal. In contrast to Drosophila TRPA1, Anopheles gambiae TRPA1 is directly and potently activated by citronellal, thereby raising the possibility that mosquito TRPA1 may be a target for developing improved repellents to reduce insect-borne diseases such as malaria (Kwon, 2010).

Two features of the citronellal responses were found to be abnormal in the trpA1¹ basiconic sensilla ab11a neurons. First, there was a higher citronellal-evoked action potential frequency than in wild-type. Second, there was a defect in deactivation in trpA1¹ ab11a neurons. The same two defective phenotypes were observed in ab11 neurons in the dGqα¹ and norpA^P24 mutants, although only the increase in the evoked responses was clearly different when testing significance by analysis of variance. These results support the conclusion that the dGqα¹, norpA^P24, and trpA1¹ mutations affect the citronellal response in an ORN in ab11 (Kwon, 2010).

The finding that there were increases in the frequency of citronellal-evoked action potentials was unexpected and raised a question as to the basis for these defects. TRPA1 is a Ca²⁺-permeable channel, and because reduced activity of Ca²⁺-activated K⁺ channels (BK channels) increases the frequency of action potential firing, it was of interest to see whether loss of TRPA1 caused reduced BK channel activity. If so, then a mutation in the gene (slowpoke, slo) encoding the fly BK channel might phenocopy the trpA1 phenotype. In support of this model, the slo^f05915 mutation caused an increase in the frequency of citronellal-evoked action potentials and impaired citronellal avoidance. Introduction of UAS-slo-RNAi in combination with either the trpA1-GAL4 or the Or83b-GAL4 resulted in a similar defect in citronellal avoidance (Kwon, 2010).

It is proposed that TRPA1 is required for activity of Slo, which in turn modulates citronellal-induced firing of action potentials. Slo might be required in many ORNs and be regulated by additional TRP channels. In support of this proposal, ab12 also responded to citronellal and displayed a higher frequency of action potentials in slo^f05915 but did not function through a Gqα/PLC/TRPA1 pathway. Knockout of a mammalian TRP channel, TRPC1, also disrupts the activity of a Ca²⁺-activated K⁺ channel (K_Ca) in salivary gland cells, and mutations affecting either TRPC1 or K_Ca result in similar defects in salivary gland secretion. Thus, a role for TRP channels in activating Ca²⁺-activated K⁺ channels might be a common but poorly appreciated general phenomenon that is evolutionarily conserved (Kwon, 2010).

The finding that loss of TRPA1 causes an increase rather than a decrease in citronellal-induced action potentials suggests that there might be a TRPA1 independent-pathway required for generating action potentials in response to citronellal. OR83b is a candidate for functioning in such a pathway, because mutation of Or83b interferes with the ability of the synthetic repellent DEET to inhibit the attraction to food odors. This study found that Or83b¹ mutant flies, or Or83b¹ in trans with a deficiency that uncovers the locus, exhibited an impairment in citronellal avoidance similar to that in trpA1 mutant flies. An Or83b¹ defect in the DART assay was not specific to citronellal, because these flies were also impaired in the response to benzaldehyde. Tested were performed to see whether the frequency of citronellal-induced action potentials was altered in Or83b¹ ab11 sensilla. In contrast to the trpA1 mutant phenotype, none of the mutant Or83b¹ ab11 neurons responded to citronellal (Kwon, 2010).

These data indicate that there are dual pathways required for the response to citronellal. OR83b is necessary for producing citronellal-induced action potentials, and a Gq/PLC/TRPA1 pathway appears to function in the modulation of action potential frequency by activating BK channels. It is suggested that an abnormally high frequency of action potentials may lead to rapid depletion of the readily releasable pools of neurotransmitter, thereby muting the citronellal response. Interestingly, a loss-of-function mutation affecting a worm BK channel also results in a behavioral phenotype—increased resistance to ethanol. Although Drosophila TRPA1 functions downstream of a Gq/PLC signaling pathway, citronellal can also directly activate TRPA1, but with low potency. Nevertheless, because Anopheles gambiae TRPA1 is also expressed in the antenna and is activated directly by citronellal with high potency, it is suggested that mosquito TRPA1 represents a new potential target for in vitro screens for volatile activators that might serve as new types of insect repellents (Kwon, 2010).

Dissection of gain control mechanisms in Drosophila mechanotransduction

Mechanoreceptor cells respond to a vast span of stimulus intensities, which they transduce into a limited response-range using a dynamic regulation of transduction gain. Weak stimuli are detected by enhancing the gain of responses through the process of active mechanical amplification. To preserve responsiveness, the gain of responses to prolonged activation is rapidly reduced through the process of adaptation. This study investigated long-term processes of mechanotransduction gain control by studying responses from single mechanoreceptor neurons in Drosophila. Mechanical stimuli were found to elicite a sustained reduction of gain that has been termed long-term adaptation. Long-term adaptation and the adaptive decay of responses during stimuli had distinct kinetics and they were independently affected by manipulations of mechanotransduction. Therefore, long-term adaptation is not associated with the reduction of response gain during stimulation. Instead, the long-term adaptation suppressed canonical features of active amplification which were the high gain of weak stimuli and the spontaneous emission of noise. In addition, depressing amplification using energy deprivation recapitulated the effects of long-term adaptation. These data suggest that long-term adaptation is mediated by suppression of active amplification. Finally, the extent of long-term adaptation matched with cytoplasmic Ca(2+) levels and dTrpA1-induced Ca(2+) elevation elicited the effects of long-term adaptation. The data suggest that mechanotransduction employs parallel adaptive mechanisms: while a rapid process exerts immediate gain reduction, long-term adjustments are achieved by attenuating active amplification. The slow adjustment of gain, manifest as diminished sensitivity, is associated with the accumulation of Ca(2+) (Chadha, 2012).

Following activation the gain in mechanoreceptor neurons is depressed for hundreds of milliseconds, and this depressing effect is generated over a similar time scale. These characteristics are in accord with findings in other mechanoreceptor neurons. The depressing effect of activation was substantial at the lower end of test-response intensities and minimally affected responses at saturation. Therefore, the temporal resolution of mechanotransduction remains high when incoming signals are at saturating levels but severely diminishes when weak stimuli follow strong ones (Chadha, 2012).

Changing the duration and the magnitude of conditioning pulses had comparable effects on long-term adaptation. Since a consistent correlation was observed between the extent of long-term adaptation and the integrated conditioning current it is postulated that the reduction of gain is determined mainly by integrating activation during a wide time window (Chadha, 2012).

The current–displacement curve during long-term adaptation was characterized by a right-shift along the displacement axis, accompanied by a reduction in slope. A right-shift in the current–displacement curve is a well described characteristic of adaptation and it typically retains the slope of the curve. Nevertheless, reductions in slope have also been reported in hair cells, mainly by application of large adapting steps. Therefore, it is possible that long-term adaptation and slow adaptation share underlying mechanisms (Chadha, 2012).

To identify the transduction process that underlies long-term adaptation the possibility was tested that it is generated by the same mechanism that causes the adaptive response decay. It was found that upon stimulation with two consecutive saturating stimuli the response recovery was nearly complete even at the shortest interval. The mechanoreceptor neuron's ability to evoke the transient phase shortly after the termination of a fully adapted response suggests that the adaptive response decay reverses rapidly. In addition, the generation of long-term adaptation and adaptive response decay take place at very different time scales; when adaptive response decay was already at a steady-state level, the effect of long-term adaptation continued to grow considerably (Chadha, 2012).

Given that adaptive response decay saturates long before long-term adaptation, it is likely that the two processes are generated in parallel. To further test the link between long-term adaptation and adaptive response decay the holding potential was changed, a manipulation that substantially affects adaptive response decay. It was found that the changes in the properties of adaptive response decay were not associated with an effect on long-term adaptation. To characterize the process at the molecular levela specific mutation was tested that affects adaptation in nompC, which is a mechanosensitive channel. The mutant, nompC⁴, demonstrated the expected rapid kinetics of adaptive response decay; however, they were not associated with corresponding changes to long-term adaptation. Therefore, both by changing the holding potential and by a specific mutation it was demonstrated that changes in the adaptive response decay are not accompanied by corresponding changes in long-term adaptation. The lack of association was also supported by the converse observation; induction of long-term adaptation using hypoxia was not accompanied by an effect on adaptive response decay (Chadha, 2012).

Altogether, these findings indicate that there are at least two separate gain adjustment processes in Drosophila mechanotransduction: the rapid decay of response amplitude during a stimulus and long-term adaptation, which adjusts mechanotransduction sensitivity to the average stimulus intensity over a wide time window. Previous studies in hair cells demonstrated that adaptation to sustained stimuli is comprised of two distinct processes with separate time courses. The time course of the slow adaptation process may reach hundreds of milliseconds suggesting that it may share a common underlying mechanism with the long-term adaptation that this study observed. It has been suggested that by determining the set point of mechanotransduction, adaptation biases it toward operating at a region mechanical instability. The interplay between adaptation adjustment mechanisms and the point of instability is thought to underlie active amplification. It is therefore possible that the long-term adaptation results from shifting away from the region of mechanical instability, thereby diminishing amplification and spontaneous noise. Alternatively, since the time constant of long-term adaptation is still slightly higher than the upper range of slow adaptation, it is possible that a separate transduction mechanism is involved. Future studies are necessary for testing whether the slow component of response decay and the long-term adaptation are linked (Chadha, 2012).

The specific effect of long-term adaptation on low-end response intensities and on the slope of the current–displacement curve are in accord with reduced amplification. A prominent feature of active amplification, the spontaneously emitted noise is affected by conditioning activation in the same manner as test-responses. To further link between long-term adaptation and active amplification suppression energy availability was limited. Limiting energy availability using hypoxia has been shown to severely and reversibly diminish auditory sensitivity and the occurrence of spontaneous noise emissions. Hypoxic conditions were defined that did not affect general response characteristics while they were sufficient for recapitulating long-term adaptation. The effects of hypoxia and preactivation were similar on both test-responses and the electrical noise, although the right-shift in the current–displacement curve was stronger during hypoxia. The stronger hypoxia effect is probably caused by the direct superposition of test pulses onto the treatment, as opposed to the partial recovery that occurs after the cessation of conditioning stimuli. In summary, there are three independent lines of evidence which indicate that long-term adaptation is mediated by active amplification suppression: preceding activation specifically affects sensitivity, activation diminishes the emission of spontaneous noise and both these effects can be mimicked by energy deprivation (Chadha, 2012).

Active amplification requires energy and generates noise and it would therefore be evolutionarily advantageous to attenuate the gain during extended periods of elevated activation. Under such conditions amplification is rendered unnecessary since the elevated background levels would mask weaker incoming stimuli. Another beneficial feature of long-term adaptation arises from the mechanism of its induction which does not require external application of mechanical forces. This type of a mechanism, which probably involves a diffusible second messenger, is able to regulate sensitivity by integrating activation levels with additional cell-signaling factors. These factors may be intracellular, such as a reaction to cellular stress, or intercellular, as in hormonal signaling. Effects of cellular messengers on mechanotransduction properties have been reported for the cAMP and PIP2 pathways (Chadha, 2012).

Calcium ions may be general modulators of mechanotransduction since they can modulate spontaneous mechanical oscillation and they are necessary for both slow and fast adaptation. Therefore, the inflow of Ca²⁺ may underlie the consistent correlation between the total charge inflow during activation and the extent of long-term adaptation. To study Ca²⁺ dynamics during mechanical responses, a method was developed for Ca²⁺ imaging from the same neurons that were characterized electrophysiologically. It was found that the dynamics of cellular Ca²⁺ levels were correlated with the time courses of long-term adaptation and dramatically slower than the kinetics of adaptive response decay. To induce cellular Ca²⁺ elevation the heat-activated channel dTrpA1 was expressed in the mechanoreceptor neurons. It was found that dTrpA1 activation was sufficient for recapitulating the effects of long-term adaptation. It is proposed that activation is integrated through the accumulation of Ca²⁺, a process which leads to suppression of active amplification through a yet unknown mechanism (Chadha, 2012).

Measurements of antennal motions and compound action potentials indicate that the auditory system of the fly actively amplifies mechanical stimuli and adapts to them. Such studies are well suited for defining the contribution of molecular components to amplification processes, because of the sensitive measurements of mechanical motions. However, these experiments measure the function of the entire hearing organ and are therefore unable to resolve response from single cells and to define mechanically induced currents. This study was able to separate between distinct transduction processes and characterize their properties using measurements of current responses from single receptor neurons. Together, the sensitive motion analysis of amplification and detailed characterization of single cell responses are expected to be a useful platform for genetic studies of transduction components (Chadha, 2012).

Converging circuits mediate temperature and shock aversive olfactory conditioning in Drosophila

Drosophila learn to avoid odors that are paired with aversive stimuli. Electric shock is a potent aversive stimulus that acts via dopamine neurons to elicit avoidance of the associated odor. While dopamine signaling has been demonstrated to mediate olfactory electric shock conditioning, it remains unclear how this pathway is involved in other types of behavioral reinforcement, such as in learned avoidance of odors paired with increased temperature. To better understand the neural mechanisms of distinct aversive reinforcement signals, an olfactory temperature conditioning assay were established comparable to olfactory electric shock conditioning. The AC neurons, which are internal thermal receptors expressing dTrpA1, are selectively required for odor-temperature but not for odor-shock memory. Furthermore, these separate sensory pathways for increased temperature and shock converge onto overlapping populations of dopamine neurons that signal aversive reinforcement. Temperature conditioning appears to require a subset of the dopamine neurons required for electric shock conditioning. It is concluded that dopamine neurons integrate different noxious signals into a general aversive reinforcement pathway (Galili, 2014).

Drosophila circadian rhythms in seminatural environments: Summer afternoon component is not an artifact and requires TrpA1 channels

Under standard laboratory conditions of rectangular light/dark cycles and constant warm temperature, Drosophila melanogaster show bursts of morning (M) and evening (E) locomotor activity and a "siesta" in the middle of the day. These M and E components have been critical for developing the neuronal dual oscillator model in which clock gene expression in key cells generates the circadian phenotype. However, under natural European summer conditions of cycling temperature and light intensity, an additional prominent afternoon (A) component that replaces the siesta is observed. This component has been described as an "artifact" of the TriKinetics locomotor monitoring system that is used by many circadian laboratories world wide. Using video recordings, this study shows that the A component is not an artifact, neither in the glass tubes used in TriKinetics monitors nor in open-field arenas. By studying various mutants in the visual and peripheral and internal thermo-sensitive pathways, it was revealed that the M component is predominantly dependent on visual input, whereas the A component requires the internal thermo-sensitive channel Transient receptor potential A1 (TrpA1). Knockdown of TrpA1 in different neuronal groups reveals that the reported expression of TrpA1 in clock neurons is unlikely to be involved in generating the summer locomotor profile, suggesting that other TrpA1 neurons are responsible for the A component. Studies of circadian rhythms under seminatural conditions therefore provide additional insights into the molecular basis of circadian entrainment that would otherwise be lost under the usual standard laboratory protocols (Green, 2015).

dTRPA1 modulates afternoon peak of activity of fruit flies Drosophila melanogaster

Circadian rhythms in Drosophila under semi-natural conditions (or SN) have received much recent attention. One of the striking differences in the behaviour of wild type flies under SN is the presence of an additional peak of activity in the middle of the day. This is referred to as the afternoon peak (A-peak) and is absent under standard laboratory regimes using gated light and temperature cues. Although previous reports identified the physical factors that contribute towards the A-peak there is no evidence for underlying molecular mechanisms or pathways that control A-peak. This study reports that the A-peak is mediated by thermosensitive TRPA1 (Transient Receptor Potential A1) ion channels as this peak is absent in TRPA1 null mutants. Further, when natural cycles of light and temperature are simulated in the lab, the amplitude of the A-peak was found to be TRPA1-dependent. Although a few circadian neurons express TRPA1, modulation of A-peak is primarily influenced by non-CRY TRPA1 expressing neurons. Hence, it is proposed that A-peak of activity observed under SN is a temperature sensitive response in flies that is elicited through TRPA1 receptor signalling (Das, 2015).

Piezo is essential for amiloride-sensitive stretch-activated mechanotransduction in larval Drosophila dorsal bipolar dendritic sensory neurons

Stretch-activated afferent neurons, such as those of mammalian muscle spindles, are essential for proprioception and motor co-ordination, but the underlying mechanisms of mechanotransduction are poorly understood. The dorsal bipolar dendritic (dbd) sensory neurons are putative stretch receptors in the Drosophila larval body wall. An in vivo protocol was developed to obtain receptor potential recordings from intact dbd neurons in response to stretch. Receptor potential changes in dbd neurons in response to stretch showed a complex, dynamic profile with similar characteristics to those previously observed for mammalian muscle spindles. These profiles were reproduced by a general in silico model of stretch-activated neurons. This in silico model predicts an essential role for a mechanosensory cation channel (MSC) in all aspects of receptor potential generation. Using pharmacological and genetic techniques, the mechanosensory channel, Piezo, was identified in this functional role in dbd neurons, with TRPA1 playing a subsidiary role. It was also shown that rat muscle spindles exhibit a ruthenium red-sensitive current, but no expression evidence was found to suggest that this corresponds to Piezo activity. In summary, this study shows that the dbd neuron is a stretch receptor and demonstrates that this neuron is a tractable model for investigating mechanisms of mechanotransduction (Suslak, 2015).

Loss of Drosophila melanogaster TRPA1 function affects "siesta" behavior but not synchronization to temperature cycles

To maintain synchrony with the environment, circadian clocks use a wide range of cycling sensory cues that provide input to the clock (zeitgebers), including environmental temperature cycles (TCs). There is some knowledge about which clock neuronal groups are important for temperature synchronization, knowledge on the temperature receptors and their signaling pathways that feed temperature information to the (neuronal) clock is lacking. Since TRPA1 is a well-known thermosensor that functions in a range of temperature-related behaviors, and it is potentially expressed in clock neurons, this study set out to test the putative role of TRPA1 in temperature synchronization of the circadian clock. Flies lacking TRPA1 are still able to synchronize their behavioral activity to TCs comparable to wild-type flies, both in 16^o C : 25^o C and 20^o C : 29^o C TCs. In addition, it was found that flies lacking TRPA1 show higher activity levels during the middle of the warm phase of 20 ^o C : 29^o C TCs, and it was show that this TRPA1-mediated repression of locomotor activity during the 'siesta' is caused by a lack of sleep. Based on these data, it is concluded that the TRPA1 channel is not required for temperature synchronization in this broad temperature range but instead is required to repress activity during the warm part of the day (Roessingh, 2015).

dTRPA1 in Non-circadian Neurons Modulates Temperature-Dependent Rhythmic Activity in Drosophila melanogaster

In fruit flies Drosophila melanogaster, environmental cycles of light and temperature are known to influence behavioral rhythms through dedicated sensory receptors. But the thermosensory pathways and molecular receptors by which thermal cycles modulate locomotor activity rhythms remain unclear. This study reports that neurons expressing warmth-activated ion channel Drosophila Transient Receptor Potential-A1 (dTRPA1) modulate distinct aspects of the rhythmic activity/rest rhythm in a light-dependent manner. Under light/dark (LD) cycles paired with constantly warm ambient conditions, flies deficient in dTRPA1 expression are unable to phase morning and evening activity bouts appropriately. Correspondingly, it was shown that electrical activity of a few neurons targeted by the dTRPA^1SH-GAL4 driver modulates temperature-dependent phasing of activity/rest rhythm under LD cycles. The expression of dTRPA1 also affects behavior responses to temperature cycles combined with constant dark (DD) or light (LL) conditions. The mid-day 'siesta' exhibited by flies under temperature cycles in DD is dependent on dTRPA1 expression in a small number of neurons that include thermosensory anterior cell neurons. Although a small subset of circadian pacemaker neurons may express dTRPA1, it was shown that CRY-negative dTRPA^1SH-GAL4 driven neurons are critical for the suppression of mid-thermophase activity, thus enabling flies to exhibit siesta. In contrast to temperature cycles in DD, under LL, dTRPA1 is not required for exhibiting siesta but is important for phasing of evening peak. These studies show that activity/rest rhythms are modulated in a temperature-dependent manner via signals from dTRPA1SH-GAL4 driven neurons. Taken together, these results emphasize the differential influence of thermoreceptors on rhythmic behavior in fruit flies in coordination with light inputs (Das, 2016).

Contribution of Drosophila TRPA1 to metabolism

Transient receptor potential (TRP) cation channels are highly conserved in humans and insects. Some of these channels are expressed in internal organs and their functions remain incompletely understood. By direct knock-in of the GAL4 gene into the trpA1 locus in Drosophila, this study identified the expression of this gene in the subesophageal ganglion (SOGs) region. In addition, the neurites present in the dorsal posterior region as well as the Insulin-like peptide 2 (ILP2)-positive neurons send signals to the SOGs. The signal is sent to the crop, which is an enlarged organ of the esophagus and functions as a storage place for food in the digestive system. To systematically investigate the role of TRPA1 in metabolism, non-targeted metabolite profiling analysis together with gas-chromatography/time-of-flight mass spectrometry, were applied with an aim to identify a wide range of primary metabolites. Distinctive metabolomic phenotypes were effectively captured, and specific metabolic dysregulation triggered by TRPA1 mutation was identified based on reconstructed metabolic network analysis. Primarily, the network analysis pinpointed the simultaneous down-regulation of intermediates in the methionine salvation pathway, in contrast to the synchronized up-regulation of a range of free fatty acids. The gene dosage-dependent dynamics of metabolite levels among wild-type, hetero- and homozygous mutants, and their coordinated metabolic modulation under multiple gene settings across five different genotypes confirmed the direct linkages of TRPA1 to metabolism (Lee, 2016).

Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila

Detecting pathogens and mounting immune responses upon infection is crucial for animal health. However, these responses come at a high metabolic price, and avoiding pathogens before infection may be advantageous. The bacterial endotoxins lipopolysaccharides (LPS) are important immune system infection cues, but it remains unknown whether animals possess sensory mechanisms to detect them prior to infection. This study shows that fruit flies display strong aversive responses to LPS and that gustatory neurons expressing Gr66a bitter receptors mediate avoidance of LPS in feeding and egg laying assays. The expression of the chemosensory cation channel dTRPA1 in these cells was found to be necessary and sufficient for LPS avoidance. Furthermore, LPS stimulates Drosophila neurons in a TRPA1-dependent manner and activates exogenous dTRPA1 channels in human cells. These findings demonstrate that flies detect bacterial endotoxins via a gustatory pathway through TRPA1 activation as conserved molecular mechanism (Soldano, 2016).

Nucleophile sensitivity of Drosophila TRPA1 underlies light-induced feeding deterrence

Solar irradiation including ultraviolet (UV) light causes tissue damage by generating reactive free radicals that can be electrophilic or nucleophilic due to unpaired electrons. Little is known about how free radicals induced by natural sunlight are rapidly detected and avoided by animals. This study found that Drosophila Transient Receptor Potential Ankyrin 1 (TRPA1), previously known only as an electrophile receptor, sensitively detects photochemically active sunlight through nucleophile sensitivity. Rapid light-dependent feeding deterrence in Drosophila was was found to be mediated only by the TRPA1(A) isoform, despite the TRPA1(A) and TRPA1(B) isoforms having similar electrophile sensitivities. Such isoform dependence re-emerges in the detection of structurally varied nucleophilic compounds and nucleophilicity-accompanying hydrogen peroxide (H₂O₂). Furthermore, these isoform-dependent mechanisms require a common set of TRPA1(A)-specific residues dispensable for electrophile detection. Collectively, TRPA1(A) rapidly responds to natural sunlight intensities through its nucleophile sensitivity as a receptor of photochemically generated radicals, leading to an acute light-induced behavioral shift in Drosophila (Du, 2016).

TRPA1 mediates sensation of the rate of temperature change in Drosophila larvae. Nat Neurosci

Avoidance of noxious ambient heat is crucial for survival. A well-known phenomenon is that animals are sensitive to the rate of temperature change. However, the cellular and molecular underpinnings through which animals sense and respond much more vigorously to fast temperature changes are unknown. Using Drosophila larvae, this study found that nociceptive rolling behavior was triggered at lower temperatures and at higher frequencies when the temperature increased rapidly. Neurons in the brain were identified that were sensitive to the speed of the temperature increase rather than just to the absolute temperature. These cellular and behavioral responses depended on the TRPA1 channel, whose activity responded to the rate of temperature increase. It is proposed that larvae use low-threshold sensors in the brain to monitor rapid temperature increases as a protective alert signal to trigger rolling behaviors, allowing fast escape before the temperature of the brain rises to dangerous levels (Luo, 2016).

H₂O₂-sensitive isoforms of Drosophila TRPA1 act in bitter-sensing gustatory neurons to promote avoidance of UV during egg-laying

Recent results have suggested that specific isoforms of Drosophila TRPA1 (dTRPA1) are UV sensitive and that their UV sensitivity is due to H₂O₂ sensitivity. This study has demonstrated that H₂O₂-sensitive dTRPA1 isoforms promote avoidance of UV when adult Drosophila females are selecting sites for egg-laying. Blind/visionless females are still capable of sensing and avoiding UV during egg-laying when intensity of UV is high yet within the range of natural sunlight. Second, such vision-independent UV avoidance was shown to be mediated by a group of bitter-sensing neurons on the proboscis that express H₂O₂-sensitive dTRPA1 isoforms. These bitter-sensing neurons exhibit dTRPA1-dependent UV sensitivity. Importantly, inhibiting activities of these bitter-sensing neurons, reducing their dTRPA1 expression, or reducing their H₂O₂-sensitivity all significantly reduced blind females' UV avoidance, whereas selectively restoring a H₂O₂-sensitive isoform of dTRPA1 in these neurons restored UV avoidance. Lastly, it was shown that expressing the red-shifted channelrhodopsin CsChrimson specifically in these bitter-sensing neurons promotes egg-laying avoidance of red light, an otherwise neutral cue for egg-laying females (Guntur, 2016).

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).

The PDZ assembled "transducisome" of microvillar photoreceptors: the TRP/TRPL problem

Two types of ion channels, TRP and TRPL, are activated upon light-absorption in rhabdomeral photoreceptor membranes of fly compound eyes. Whereas TRP is associated with other signaling proteins into a multiprotein complex (transducisome), the molecular organization of TRPL is discussed controversely. This study analysed the TRPL content of blowfly rhabdomeral membranes and investigated by co-immunoprecipitation studies whether or not TRPL is part of the transducisome. Compared to TRP there are at least ten times less TRPL molecules present in the rhabdomeral membrane. A small fraction of the total TRPL present co-immunoprecipitates with other proteins of the transducisome and vice versa. These data suggest that a significant fraction of TRPL is not incorporated into the transducisome. This fraction may either form independent ion channels or bind to the transducisome transiently (Paulsen, 2016).

REFERENCES

Search PubMed for articles about Drosophila Transient receptor potential A1

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date revised: 15 December 2023

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