Regulation of thermoregulatory behavior by commensal bacteria in Drosophila.

Commensal bacteria affect many aspects of host physiology. In this study, we focused on the role of commensal bacteria in the thermoregulatory behavior of Drosophila melanogaster. We demonstrated that the elimination of commensal bacteria caused an increase in the preferred temperature of Drosophila third-instar larvae without affecting the activity of transient receptor potential ankyrin 1 (TRPA1)-expressing thermosensitive neurons. We isolated eight bacterial strains from the gut and culture medium of conventionally reared larvae and found that the preferred temperature of the larvae was decreased by mono-association with Lactobacillus plantarum or Corynebacterium nuruki. Mono-association with these bacteria did not affect the indices of energy metabolism such as ATP and glucose levels of larvae, which are closely linked to thermoregulation in animals. Thus, we show a novel role for commensal bacteria in host thermoregulation and identify two bacterial species that affect thermoregulatory behavior in Drosophila.

Embryonic thermosensitive TRPA1 determines transgenerational diapause phenotype of the silkworm, Bombyx mori.

In the bivoltine strain of the silkworm, Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother; however, its molecular mechanisms are largely unknown. Here, we show that the Bombyx TRPA1 ortholog (BmTrpA1) acts as a thermosensitive transient receptor potential (TRP) channel that is activated at temperatures above ∼ 21 °C and affects the induction of diapause in progeny. In addition, we show that embryonic RNAi of BmTrpA1 affects diapause hormone release during pupal-adult development. This study identifying a thermosensitive TRP channel that acts as a molecular switch for a relatively long-term predictive adaptive response by inducing an alternative phenotype to seasonal polyphenism is unique.

Functional roles of TRPV1 and TRPV4 in control of lower urinary tract activity: dual analysis of behavior and reflex during the micturition cycle.

The present study used a dual analysis of voiding behavior and reflex micturition to examine lower urinary tract function in transient receptor potential vanilloid (TRPV)1 knockout (KO) mice and TRPV4 KO mice. In metabolic cage experiments conducted under conscious conditions (i.e., voluntary voiding behavior), TRPV4 KO mice showed a markedly higher voiding frequency (VF; 19.3 ± 1.2 times/day) and a smaller urine volume/voiding (UVV; 114 ± 9 µl) compared with wild-type (WT) littermates (VF: 5.2 ± 0.5 times/day and UVV: 380 ± 34 µl). Meanwhile, TRPV1 KO mice showed a similar VF to WT littermates (6.8 ± 0.5 times/day) with a significantly smaller UVV (276 ± 20 µl). Water intake among these genotypes was the same, but TRPV4 KO mice had a larger urine output than the other two groups. In cystometrogram experiments conducted in decerebrate unanesthetized mice (i.e., reflex micturition response), no differences between the three groups were found in any cystometrogram variables, including voided volume, volume threshold for inducing micturition contraction, maximal voiding pressure, and bladder compliance. However, both TRPV1 KO and TRPV4 KO mice showed a significant number of nonvoiding bladder contractions (NVCs; 3.5 ± 0.9 and 2.8 ± 0.7 contractions, respectively) before each voiding, whereas WT mice showed virtually no NVCs. These results suggest that in the reflex micturition circuit, a lack of either channel is involved in NVCs during bladder filling, whereas in the forebrain, it is involved in the early timing of urine release, possibly in the conscious response to the bladder instability.

Redox signal-mediated sensitization of transient receptor potential melastatin 2 (TRPM2) to temperature affects macrophage functions.

The ability to sense temperature is essential for organism survival and efficient metabolism. Body temperatures profoundly affect many physiological functions, including immunity. Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive, Ca(2+)-permeable cation channel expressed in a wide range of immunocytes. TRPM2 is activated by adenosine diphosphate ribose and hydrogen peroxide (H(2)O(2)), although the activation mechanism by H(2)O(2) is not well understood. Here we report a unique activation mechanism in which H(2)O(2) lowers the temperature threshold for TRPM2 activation, termed "sensitization," through Met oxidation and adenosine diphosphate ribose production. This sensitization is completely abolished by a single mutation at Met-214, indicating that the temperature threshold of TRPM2 activation is regulated by redox signals that enable channel activity at physiological body temperatures. Loss of TRPM2 attenuates zymosan-evoked macrophage functions, including cytokine release and fever-enhanced phagocytic activity. These findings suggest that redox signals sensitize TRPM2 downstream of NADPH oxidase activity and make TRPM2 active at physiological body temperature, leading to increased cytosolic Ca(2+) concentrations. Our results suggest that TRPM2 sensitization plays important roles in macrophage functions.

Ambient temperature affects the temperature threshold for TRPM8 activation through interaction of phosphatidylinositol 4,5-bisphosphate.

Cold sensation is an important and fundamental sense for animals and it is known to be affected by ambient temperature. Transient Receptor Potential Melastatin 8 (TRPM8), a nonselective cation channel expressed in a subset of peripheral afferent fibers, acts as a cold sensor, having an activation threshold of ∼28°C. Although the cold temperature threshold of TRPM8 is affected by menthol or pH, ambient temperature has not been reported to affect it. Because the cold temperature threshold was thought to be unchanged by alterations in ambient temperature, the relativity of temperature sensing in different ambient temperatures could not be understood at the level of molecular function of thermosensitive TRP channels. Here, we show that ambient temperature changed the temperature threshold for activation of human and rat TRPM8 in a heterologous expression system and cold responses in mouse DRG neurons. Moreover, reducing the level of cellular phosphatidylinositol 4,5-bisphosphate (PIP2) attenuated changes in the cold temperature threshold after alterations in ambient temperature. A single amino acid mutation at position 1008 in the C terminus of TRPM8 (arginine to glutamine) also attenuated changes in the cold temperature threshold induced by ambient temperature. These findings suggest that ambient temperature does affect the temperature threshold for TRPM8 activation through interaction of PIP2.

Activation of an essential calcium signaling pathway in Saccharomyces cerevisiae by Kch1 and Kch2, putative low-affinity potassium transporters.

In the budding yeast Saccharomyces cerevisiae, mating pheromones activate a high-affinity Ca(2+) influx system (HACS) that activates calcineurin and is essential for cell survival. Here we identify extracellular K(+) and a homologous pair of transmembrane proteins, Kch1 and Kch2 (Prm6), as necessary components of the HACS activation mechanism. Expression of Kch1 and especially Kch2 was strongly induced during the response to mating pheromones. When forcibly overexpressed, Kch1 and Kch2 localized to the plasma membrane and activated HACS in a fashion that depended on extracellular K(+) but not pheromones. They also promoted growth of trk1 trk2 mutant cells in low K(+) environments, suggesting they promote K(+) uptake. Voltage-clamp recordings of protoplasts revealed diminished inward K(+) currents in kch1 kch2 double-mutant cells relative to the wild type. Conversely, heterologous expression of Kch1 in HEK293T cells caused the appearance of inwardly rectifying K(+) currents. Collectively, these findings suggest that Kch1 and Kch2 directly promote K(+) influx and that HACS may electrochemically respond to K(+) influx in much the same way as the homologous voltage-gated Ca(2+) channels in most animal cell types.

G protein-coupled receptor-based thermosensation determines temperature acclimatization of Caenorhabditis elegans.

Animals must sense and acclimatize to environmental temperatures for survival, yet their thermosensing mechanisms other than transient receptor potential (TRP) channels remain poorly understood. We identify a trimeric G protein-coupled receptor (GPCR), SRH-40, which confers thermosensitivity in sensory neurons regulating temperature acclimatization in Caenorhabditis elegans. Systematic knockdown of 1000 GPCRs by RNAi reveals GPCRs involved in temperature acclimatization, among which srh-40 is highly expressed in the ADL sensory neuron, a temperature-responsive chemosensory neuron, where TRP channels act as accessorial thermoreceptors. In vivo Ca2+ imaging demonstrates that an srh-40 mutation reduced the temperature sensitivity of ADL, resulting in supranormal temperature acclimatization. Ectopically expressing SRH-40 in a non-warmth-sensing gustatory neuron confers temperature responses. Moreover, temperature-dependent SRH-40 activation is reconstituted in Drosophila S2R+ cells. Overall, SRH-40 may be involved in thermosensory signaling underlying temperature acclimatization. We propose a dual thermosensing machinery through a GPCR and TRP channels in a single sensory neuron.

Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice.

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4(-/-) mice do not have normal endothelial cell responses to flow, such as influx of Ca(2+) and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4(-/-) mice. Furthermore, P2rx4(-/-) mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4(-/-) mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.

Avoidance of thiazoline compound depends on multiple sensory pathways mediated by TrpA1 and ORs in Drosophila.

Transient receptor potential (TRP) channels are primary sensory molecules in animals and are involved in detecting a diverse range of physical and chemical cues in the environments. Considering the crucial role of TRPA1 channels in nocifensive behaviors and aversive responses across various insect species, activators of TRPA1 are promising candidates for insect pest control. In this study, we demonstrate that 2-methylthiazoline (2MT), an artificial volatile thiazoline compound originally identified as a stimulant for mouse TRPA1, can be utilized as a novel repellent for fruit flies, Drosophila melanogaster. We observed that 2MT induced strong, dose-dependent avoidance behaviors in adult males, regardless of their feeding states, as well as egg laying behavior in females. These aversive responses were mediated by contact chemosensation via TrpA1 and olfaction via odorant receptors. Knocking down TrpA1 revealed the essential roles of bitter taste neurons and nociceptive neurons in the legs and labellum. Furthermore, among five isoforms, TrpA1-C and TrpA1-D exclusively contributed to the aversiveness of 2MT. We also discovered that these isoforms were directly activated by 2MT through covalent modification of evolutionarily conserved cysteine residues. In conclusion, we have identified 2MT as a stimulant for multiple sensory pathways, triggering aversive behaviors in fruit flies. We propose that 2MT and related chemicals may serve as potential resources for developing novel insect repellents.

Importance of transient receptor potential vanilloid 4 (TRPV4) in epidermal barrier function in human skin keratinocytes.

The state of the skin changes drastically depending on the ambient temperature. Skin epidermal keratinocytes express thermosensitive transient receptor potential vanilloid (TRPV) cation channels, TRPV3 and TRPV4. These multimodal receptors are activated by various kinds of chemical and physical stimuli, including warm temperatures (>30°C). It has been suggested that TRPV4 is involved in cell-cell junction maturation; however, the effect of temperature fluctuations on TRPV4-dependent barrier homeostasis is unclear. In the present study, we demonstrated that activation of TRPV4 was crucial for barrier formation and recovery, both of which were critical for the prevention of excess dehydration of human skin keratinocytes. TRPV4 activation by physiological skin temperature (33°C), GSK1016790A or 4α-PDD allowed influx of Ca(2+) from extracellular spaces which promoted cell-cell junction development. These changes resulted in augmentation of intercellular barrier integrity in vitro and ex vivo. TRPV4 disruption reduced the increase in trans-epidermal resistance and increased intercellular permeation after a Ca(2+) switch. Furthermore, barrier recovery after the disruption of the stratum corneum was accelerated by the activation of TRPV4 either by warm temperature or a chemical activator. Our results suggest that physiological skin temperatures play important roles in cell-cell junction and skin barrier homeostasis through TRPV4 activation.

Endocannabinoids produced in photoreceptor cells in response to light activate Drosophila TRP channels.

Drosophila phototransduction is a model for signaling cascades that culminate in the activation of transient receptor potential (TRP) cation channels. TRP and TRPL are the canonical TRP (TRPC) channels that are regulated by light stimulation of rhodopsin and engagement of Gαq and phospholipase Cß (PLC). Lipid metabolite(s) generated downstream of PLC are essential for the activation of the TRPC channels in photoreceptor cells. We sought to identify the key lipids produced subsequent to PLC stimulation that contribute to channel activation. Here, using genetics, lipid analysis, and Ca2+ imaging, we found that light increased the amount of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG), in vivo. The increase in 2-LG amounts depended on the PLC and diacylglycerol lipase encoded by norpA and inaE, respectively. This endocannabinoid facilitated TRPC-dependent Ca2+ influx in a heterologous expression system and in dissociated ommatidia from compound eyes. Moreover, 2-LG and mechanical stimulation cooperatively activated TRPC channels in ommatidia. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.

Honey bee thermal/chemical sensor, AmHsTRPA, reveals neofunctionalization and loss of transient receptor potential channel genes.

Insects are relatively small heterothermic animals, thus they are highly susceptible to changes in ambient temperature. However, a group of honey bees is able to maintain the brood nest temperature between 32°C and 36°C by either cooling or heating the nest. Nevertheless, how honey bees sense the ambient temperature is not known. We identified a honey bee Hymenoptera-specific transient receptor potential A (HsTRPA) channel (AmHsTRPA), which is activated by heat with an apparent threshold temperature of 34°C and insect antifeedants such as camphor in vitro. AmHsTRPA is expressed in the antennal flagellum, and ablation of the antennal flagella and injection of AmHsTRPA inhibitors impair warmth avoidance of honey bees. Gustatory responses of honey bees to sucrose are suppressed by noxious heat and insect antifeedants, but are relieved in the presence of AmHsTRPA inhibitors. These results suggest that AmHsTRPA may function as a thermal/chemical sensor in vivo. As shown previously, Hymenoptera has lost the ancient chemical sensor TRPA1; however, AmHsTRPA is able to complement the function of Drosophila melanogaster TRPA1. These results demonstrate that HsTRPA, originally arisen by the duplication of Water witch, has acquired thermal- and chemical-responsive properties, which has resulted in the loss of ancient TRPA1. Thus, this is an example of neofunctionalization of the duplicated ion channel gene followed by the loss of the functionally equivalent ancient gene.

The TRPV4 channel contributes to intercellular junction formation in keratinocytes.

Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca(2+) dynamics. We found that TRPV4 interacts with beta-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca(2+)-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca(2+) increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin.

A 3.5-nm structure of rat TRPV4 cation channel revealed by Zernike phase-contrast cryoelectron microscopy.

The transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel responsive to various stimuli including cell swelling, warm temperatures (27-35 degrees C), and chemical compounds such as phorbol ester derivatives. Here we report the three-dimensional structure of full-length rat TRPV4 purified from baculovirus-infected Sf9 cells. Hexahistidine-tagged rat TRPV4 (His-rTRPV4) was solubilized with detergent and purified through affinity chromatography and size-exclusion chromatography. Chemical cross-linking analysis revealed that detergent-solubilized His-rTRPV4 was a tetramer. The 3.5-nm structure of rat TRPV4 was determined by cryoelectron microscopy using single-particle reconstruction from Zernike phase-contrast images. The overall structure comprises two distinct regions; a larger dense component, likely corresponding to the cytoplasmic N- and C-terminal regions, and a smaller component corresponding to the transmembrane region.

Activation of polycystic kidney disease-2-like 1 (PKD2L1)-PKD1L3 complex by acid in mouse taste cells.

Five basic tastes (bitter, sweet, umami, salty, and sour) are detected in the four taste areas where taste buds reside. Although molecular mechanisms for detecting bitter, sweet, and umami have been well clarified, those for sour and salty remain poorly understood. Several channels including acid-sensing ion channels have been proposed as candidate sour receptors, but they do not encompass all sour-sensing abilities in vivo. We recently reported a novel candidate for sour sensing, the polycystic kidney disease-2-like 1 (PKD2L1)-PKD1L3 channel complex. This channel is not a traditional ligand-gated channel and is gated open only after removal of an acid stimulus, called an off response. Here we show that off responses upon acid stimulus are clearly observed in native taste cells from circumvallate, but not fungiform papillae, of glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice, from which Type III taste cells can be visualized, using Ca(2+) imaging and patch clamp methods. Off responses were detected in most cells where PKD2L1 immunoreactivity was observed. Interestingly, the pH threshold for acid-evoked intracellular Ca(2+) increase was around 5.0, a value much higher than that observed in HEK293 cells expressing the PKD2L1-PKD1L3 complex. Thus, PKD2L1-PKD1L3-mediated acid-evoked off responses occurred both in HEK293 cells and in native taste cells, suggesting the involvement of the PKD2L1-PKD1L3 complex in acid sensing in vivo.

Intracellular alkalization causes pain sensation through activation of TRPA1 in mice.

Vertebrate cells require a very narrow pH range for survival. Cells accordingly possess sensory and defense mechanisms for situations where the pH deviates from the viable range. Although the monitoring of acidic pH by sensory neurons has been attributed to several ion channels, including transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs), the mechanisms by which these cells detect alkaline pH are not well understood. Here, using Ca2+ imaging and patch-clamp recording, we showed that alkaline pH activated transient receptor potential cation channel, subfamily A, member 1 (TRPA1) and that activation of this ion channel was involved in nociception. In addition, intracellular alkalization activated TRPA1 at the whole-cell level, and single-channel openings were observed in the inside-out configuration, indicating that alkaline pH activated TRPA1 from the inside. Analyses of mutants suggested that the two N-terminal cysteine residues in TRPA1 were involved in activation by intracellular alkalization. Furthermore, intraplantar injection of ammonium chloride into the mouse hind paw caused pain-related behaviors that were not observed in TRPA1-deficient mice. These results suggest that alkaline pH causes pain sensation through activation of TRPA1 and may provide a molecular explanation for some of the human alkaline pH-related sensory disorders whose mechanisms are largely unknown.

Temperature and Sweet Taste Integration in Drosophila.

Sugar-containing foods offered at cooler temperatures tend to be less appealing to many animals. However, the mechanism through which the gustatory system senses thermal input and integrates temperature and chemical signals to produce a given behavioral output is poorly understood. To study this fundamental problem, we used the fly, Drosophila melanogaster. We found that the palatability of sucrose is strongly reduced by modest cooling. Using Ca2+ imaging and electrophysiological recordings, we demonstrate that bitter gustatory receptor neurons (GRNs) and mechanosensory neurons (MSNs) are activated by slight cooling, although sugar neurons are insensitive to the same mild stimulus. We found that a rhodopsin, Rh6, is expressed and required in bitter GRNs for cool-induced suppression of sugar appeal. Our findings reveal that the palatability of sugary food is reduced by slightly cool temperatures through different sets of thermally activated neurons, one of which depends on a rhodopsin (Rh6) for cool sensation.

Increased TRPV4 expression in non-myelinating Schwann cells is associated with demyelination after sciatic nerve injury.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective calcium-permeable cation channel that is widely expressed and activated in various neurons and glial cells in the nervous system. Schwann cells (SCs) are primary glia cells that wrap around axons to form the myelin sheath in the peripheral nervous system. However, whether TRPV4 is expressed and functions in SCs is unclear. Here, we demonstrate functional expression of TRPV4 in mouse SCs and investigated its physiological significance. Deletion of TRPV4 did not affect normal myelin development for SCs in sciatic nerves in mice. However, after sciatic nerve cut injury, TRPV4 expression levels were remarkably increased in SCs following nerve demyelination. Ablation of TRPV4 expression impaired the demyelinating process after nerve injury, resulting in delayed remyelination and functional recovery of sciatic nerves. These results suggest that local activation of TRPV4 could be an attractive pharmacological target for therapeutic intervention after peripheral nerve injury.

OSM-9 and OCR-2 TRPV channels are accessorial warm receptors in Caenorhabditis elegans temperature acclimatisation.

Caenorhabditis elegans (C. elegans) exhibits cold tolerance and temperature acclimatisation regulated by a small number of head sensory neurons, such as the ADL temperature-sensing neurons that express three transient receptor potential vanilloid (TRPV) channel subunits, OSM-9, OCR-2, and OCR-1. Here, we show that an OSM-9/OCR-2 regulates temperature acclimatisation and acts as an accessorial warmth-sensing receptor in ADL neurons. Caenorhabditis elegans TRPV channel mutants showed abnormal temperature acclimatisation. Ectopic expression of OSM-9 and OCR-2 in non-warming-responsive gustatory neurons in C. elegans and Xenopus oocytes revealed that OSM-9 and OCR-2 cooperatively responded to warming; however, neither TRPV subunit alone was responsive to warming. A warming-induced OSM-9/OCR-2-mediated current was detectable in Xenopus oocytes, yet ADL in osm-9 ocr-2 double mutant responds to warming; therefore, an OSM-9/OCR-2 TRPV channel and as yet unidentified temperature receptor might coordinate transmission of temperature signalling in ADL temperature-sensing neurons. This study demonstrates direct sensation of warming by TRPV channels in C. elegans.

Drosophila painless is a Ca2+-requiring channel activated by noxious heat.

Thermal changes activate some members of the transient receptor potential (TRP) ion channel super family. They are primary sensors for detecting environmental temperatures. The Drosophila TRP channel Painless is believed responsible for avoidance of noxious heat because painless mutant flies display defects in heat sensing. However, no studies have proven its heat responsiveness. We show that Painless expressed in human embryonic kidney-derived 293 (HEK293) cells is a noxious heat-activated, Ca(2+)-permeable channel, and the function is mostly dependent on Ca(2+). In Ca(2+)-imaging, Painless mediated a robust intracellular Ca(2+) (Ca(2+)(i)) increase during heating, and it showed heat-evoked inward currents in whole-cell patch-clamp mode. Ca(2+) permeability was much higher than that of other cations. Heat-evoked currents were negligible in the absence of extracellular Ca(2+) (Ca(2+)(o)) and Ca(2+)(i), whereas 200 nm Ca(2+)(i) enabled heat activation of Painless. Activation kinetics were significantly accelerated in the presence of Ca(2+)(i). The temperature threshold for Painless activation was 42.6 degrees C in the presence of Ca(2+)(i), whereas the threshold was significantly increased to 44.1 degrees C when only Ca(2+)(o) was present. Temperature thresholds were further reduced after repetitive heating in a Ca(2+)-dependent manner. Ca(2+)-dependent heat activation of Painless was observed at the single-channel level in excised membranes. We found that a Ca(2+)-regulatory site is located in the N-terminal region of Painless. Painless-expressing HEK293 cells were insensitive to various thermosensitive TRP channel activators including allyl isothiocyanate, whereas mammalian TRPA1 inhibitors, ruthenium red, and camphor, reversibly blocked heat activation of Painless. Our results demonstrate that Painless is a direct sensor for noxious heat in Drosophila.