a-temperature-gradient-assay-to-determine-thermal-preferences

A Temperature Gradient Assay to Determine Thermal Preferences of <em>Drosophila</em> Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

National Institutes of Natural Sciences

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.

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

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.

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

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) evoked Ca(2+) influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca(2+) concentration and ATP release. Stretch stimulation evoked intracellular Ca(2+) increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca(2+) increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca(2+) increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4alpha-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca(2+). These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.

The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures.

Sensing environmental temperature is one of the most important fundamental functions of the living things on the earth. Recently, it has been revealed that several members of the TRP ion channel super family are activated by temperature changes. A number of reports clearly demonstrate that thermal activation of these thermosensitive TRP channels contributes to various temperature-dependent responses in vivo, such as thermosensation, thermotaxis, and the regulation of cellular/tissue functions at physiological body temperature. Nine TRP channels have been reported to respond to a physiological range of temperatures in mammals. TRPV1 and TRPV2 expressed in nociceptive neurons are activated by heat (> 43 degrees C and > 52 degrees C, respectively), and TRPV1-null mice show defects in sensing noxious heat. TRPV3 and TRPV4 are predominantly expressed in skin keratinocytes rather than in sensory neurons, and the gene knock-out of each channel causes abnormal thermotaxis in vivo. TRPM8, which senses cold temperatures (< 27 degrees C), is expressed in nociceptive and non-nociceptive neurons and its loss impairs cold sensitivity. TRPA1 is expressed in nociceptive neurons and acts as a sensor for various harmful stimuli, whereas its responsiveness to noxious cold stimuli is controversial even after the analysis of mice lacking the channel. Other thermoTRPs, TRPM2, TRPM4, and TRPM5 are not expressed in sensory neurons, and are reportedly involved in several functions at physiological body temperatures including insulin secretion, taste sensation, and immune response. In this review, I summarize the molecular mechanisms of thermosensation in mammals by focusing on thermosensitive TRP channels.

[Molecular mechanisms underlying thermosensation in mammals].

Transient receptor potential V3 (TRPV3) and TRPV4 are heat-activated cation channels expressed in keratinocytes. It has been proposed that heat-activation of TRPV3 and/or TRPV4 in the skin may release diffusible molecules which would then activate termini of neighboring dorsal root ganglion (DRG) neurons. Here we show that adenosine triphosphate (ATP) is such a candidate molecule released from keratinocytes upon heating in the co-culture systems. Using TRPV1-deficient DRG neurons, we found that increase in cytosolic Ca(2+)-concentration in DRG neurons upon heating was observed only when neurons were co-cultured with keratinocytes, and this increase was blocked by P2 purinoreceptor antagonists, PPADS and suramin. In a co-culture of keratinocytes with HEK293 cells (transfected with P2X(2) cDNA to serve as a bio-sensor), we observed that heat-activated keratinocytes secretes ATP, and that ATP release is compromised in keratinocytes from TRPV3-deficient mice. This study provides evidence that ATP is a messenger molecule for mainly TRPV3-mediated thermotransduction in skin.

TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP.

TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts.

Evolutionary conservation and changes in insect TRP channels.

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.

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

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.

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

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.

The TRPV4 channel contributes to intercellular junction formation in keratinocytes.

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.

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

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.

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

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.

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

Transient receptor potential (TRP) channels play important roles in thermal, chemical, and mechanical sensation in various tissues. In this study, we investigated the differences in urothelial TRP channels between normal urothelial cells and bladder cancer cells. TRPV2 and TRPM7 expression levels and TRPV2 activator-induced intracellular Ca(2+) increases were significantly higher, whereas TRPV4 expression and TRPV4 activator-induced intracellular Ca(2+) increases were significantly lower in mouse bladder cancer (MBT-2) cells compared to normal mouse urothelial cells. The proliferation rate of MBT-2 cells overexpressing dominant-negative TRPV2 was significantly increased. In contrast, treatment with TRPV2 activators significantly decreased the proliferation rate. TRPM7-overexpressing MBT-2 cells proliferated more slowly, as compared to mock-transfected cells. Moreover, expression of dominant-negative TRPV2 significantly decreased plasma membrane Ca(2+) permeability of MBT-2 cells as compared to that in mock-transfected cells. Increases in the expression of TRPV2 mRNA, immunoreactivity, and TRPV2 activator-induced intracellular Ca(2+) were also observed in T24 human bladder cancer cells. These results suggested that TRPV2 and TRPM7 were functionally expressed in bladder cancer cells and served as negative regulators of bladder cancer cell proliferation, most likely to prevent excess mechanical stresses. 

Article	Year
Drosophila painless is a Ca2+-requiring channel activated by noxious heat. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 18829951	2008
Intracellular alkalization causes pain sensation through activation of TRPA1 in mice. The Journal of clinical investigation\| PubMed ID: 19033673	2008
The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures. The Journal of biological chemistry\| PubMed ID: 19531473	2009
[Molecular mechanisms underlying thermosensation in mammals]. Brain and nerve = Shinkei kenkyu no shinpo\| PubMed ID: 19618865	2009
TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP. Pflugers Archiv : European journal of physiology\| PubMed ID: 19669158	2009
Evolutionary conservation and changes in insect TRP channels. BMC evolutionary biology\| PubMed ID: 19740447	2009
A 3.5-nm structure of rat TRPV4 cation channel revealed by Zernike phase-contrast cryoelectron microscopy. The Journal of biological chemistry\| PubMed ID: 20044482	2010
Activation of polycystic kidney disease-2-like 1 (PKD2L1)-PKD1L3 complex by acid in mouse taste cells. The Journal of biological chemistry\| PubMed ID: 20406802	2010
The TRPV4 channel contributes to intercellular junction formation in keratinocytes. The Journal of biological chemistry\| PubMed ID: 20413591	2010
Redox signal-mediated sensitization of transient receptor potential melastatin 2 (TRPM2) to temperature affects macrophage functions. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22493272	2012
Ambient temperature affects the temperature threshold for TRPM8 activation through interaction of phosphatidylinositol 4,5-bisphosphate. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 23554496	2013
Embryonic thermosensitive TRPA1 determines transgenerational diapause phenotype of the silkworm, Bombyx mori. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24639527	2014
Potential role of transient receptor potential (TRP) channels in bladder cancer cells. The journal of physiological sciences : JPS\| PubMed ID: 24849279	2014

Takaaki Sokabe

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