The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation. The transient receptor potential (TRP)1 superfamily of cation channels consists of a large number of recently identified molecules that share sequence homology with the Drosophila protein named after a phototransduction mutant called trp. According to sequence similarities, the TRP channels are further divided into subfamilies, such as TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) (see reviews in Refs. 1 and 2). These channels are involved in diverse cellular functions including receptor and store-operated Ca 2ϩ entry (3), Ca 2ϩ transport (4, 5), trace metal detection (6), and temperature (7-9) and osmolality (10, 11) sensations. The activation mechanisms for most of the TRP channels remain to be elucidated. Specific ligands have been found for TRPC3, TRPC6, TRPC7, TRPV1, TRPV4, TRPM2, TRPM4, TRPM5, TRPM7, and TRPM8. These include endogenous substances, such as lipids (diacylglycerol (12), anandamide (13, 14), and phosphatidylinositol 4,5-bisphosphate (15)), nucleotides (ADP-ribose (16) (23,24), 2APB was soon found to directly block native store-operated channels (25-27), sarco/ endoplasmic reticulum Ca 2ϩ -ATPase pumps (28), mitochondrial permeability transition pore (29), and a few other ion channels (30). The mechanism of action for 2APB is likely to be complex. In addition to inhibition, low concentrations of 2APB enhanced the activity of store-operated channels (26). At greater than 50 M, 2APB activated a Ca 2ϩ -permeable nonselective cation channel with a 50-picosiemens single channel conductance and very low open probability in rat basophilic leukemia cells (31).2APB has been perceived as a general inhibitor of TRP channels (1). However, except for TRPC3 (24,32), the effects of this drug...

Transient receptor potential channels are involved in sensing chemical and physical changes inside and outside of cells. TRPV3 is highly expressed in skin keratinocytes, where it forms a nonselective cation channel activated by hot temperatures in the innocuous and noxious range. The channel has also been implicated in flavor sensation in oral and nasal cavities as well as being a molecular target of some allergens and skin sensitizers. TRPV3 is unique in that its activity is sensitized upon repetitive stimulations. -mediated inhibition and greatly facilitated the activation of TRPV3. We conclude that Ca 2؉ inhibits TRPV3 from both the extracellular and intracellular sides. The inhibition is sequentially reduced, appearing as sensitization to repetitive stimulations. Members of the transient receptor potential (TRP)4 superfamily of cation channels have been recognized to play important roles in sensing various environmental changes inside and outside of cells as well as the whole organisms (1). In mammals, temperature sensing is thought to be accomplished through concerted actions of a minimum of six TRP channels, i.e. TRPA1, -M8, -V4, -V3, -V1, and -V2, each covering a defined temperature range from below 17°C to above 52°C (2, 3). However, the debate remains whether some of these channels, e.g. TRPA1, are really temperature-sensitive (4). In addition, TRPM2, -M4, and -M5 have shown temperature sensing in the presence of second messenger cofactors, such as ADP-ribose and Ca 2ϩ (5, 6). Although some of the thermosensitive TRP channels are clearly expressed and functional in sensory neurons, indicative of their actions in primary afferents, others have been localized in the non-nervous tissues, for example, TRPV3 and -V4 are expressed in skin keratinocytes (7,8) and TRPV3 is, in addition, expressed in the epithelium of tongue and nose (9). The TRPV3 null mice showed some deficits in sensing hot temperatures in the innocuous and noxious range but no other obvious sensory impairment (10). On the other hand, constitutively active mutations of TRPV3 have been linked to hair loss and atopic dermatitis-like skin lesions in rodents (11,12).In addition to temperature, the thermosensitive TRP channels are activated by a large number of structurally unrelated chemical ligands from exogenous as well as endogenous sources (13). This polymodal nature has become a common feature of the TRP channel family, implicating that multiple mechanisms and external stimuli may be involved in the activation and regulation of these channels. TRPV3 was first shown to be activated by 2-aminoethoxydiphenyl borate (2APB), a synthetic compound known to inhibit inositol 1,4,5-trisphosphate receptors and store-operated channels as well as many TRP channels (14, 15). It was soon discovered that a number of natural anti-irritants and flavor enhancers such as camphor, carvacrol, thymol, and eugenol, also use TRPV3 as one of their targets (9, 10). More importantly, cell signaling events leading to the activation of phospholipase C, phosphorylation by...

Transient receptor potential vanilloid (TRPV) channels are polymodal detectors of multiple environmental factors, including temperature, pH, and pressure. Inflammatory mediators enhance TRPV function through multiple signaling pathways. The lipoxygenase and epoxygenase products of arachidonic acid (AA) metabolism have been shown to directly activate TRPV1 and TRPV4, respectively. TRPV3 is a thermosensitive channel with an intermediate temperature threshold of 31-39 degrees C. We have previously shown that TRPV3 is activated by 2-aminoethoxydiphenyl borate (2APB). Here we show that AA and other unsaturated fatty acids directly potentiate 2APB-induced responses of TRPV3 expressed in HEK293 cells, Xenopus oocytes, and mouse keratinocytes. The AA-induced potentiation is observed in intracellular Ca2+ measurement, whole-cell and two-electrode voltage clamp studies, as well as single channel recordings of excised inside-out and outside-out patches. The fatty acid-induced potentiation is not blocked by inhibitors of protein kinase C and thus differs from that induced by the kinase. The potentiation does not require AA metabolism but is rather mimicked by non-metabolizable analogs of AA. These results suggest a novel mechanism regulating the TRPV3 response to inflammation, which differs from TRPV1 and TRPV4, and involves a direct action of free fatty acids on the channel.

Transient receptor potential vanilloid 1 (TRPV1), or vanilloid receptor 1, is the founding member of the vanilloid type of TRP superfamily of nonselective cation channels. TRPV1 is activated by noxious heat, acid, and alkaloid irritants as well as several endogenous ligands and is sensitized by inflammatory factors, thereby serving important functions in detecting noxious stimuli in the sensory system and pathological states in different parts of the body. Whereas numerous studies have been carried out using the rat and human TRPV1 cDNA, the mouse TRPV1 cDNA has not been characterized. Here, we report molecular cloning of two TRPV1 cDNA variants from dorsal root ganglia of C57BL/6 mice. The deduced proteins are designated TRPV1␣ and TRPV1␤ and contain 839 and 829 amino acids, respectively. TRPV1␤ arises from an alternative intron recognition signal within exon 7 of the trpv1 gene. We found a predominant expression of TRPV1␣ in many tissues and significant expression of TRPV1␤ in dorsal root ganglia, skin, stomach, and tongue. When expressed in HEK 293 cells or Xenopus oocytes, TRPV1␣ formed a Ca 2؉ -permeable channel activated by ligands known to stimulate TRPV1. TRPV1␤ was not functional by itself but its coexpression inhibited the function of TRPV1␣. Furthermore, although both isoforms were synthesized at a similar rate, less TRPV1␤ than TRPV1␣ protein was found in cells and on the cell surface, indicating that the ␤ isoform is highly unstable. Our data suggest that TRPV1␤ is a naturally occurring dominant-negative regulator of the responses of sensory neurons to noxious stimuli. Homologues of Drosophila transient receptor potential (TRP)1 protein form a rapidly growing family of non-selective cation channels known as the TRP superfamily. The TRP channels are involved in a large variety of cellular functions including receptor and store-operated Ca 2ϩ entry (1, 2), Ca 2ϩ transport (3, 4), temperature sensation (5, 6), and trace metal detection (7). The temperature-sensing TRP channels consist of at least 6 members with temperature thresholds ranging from as low as Ͻ17°C for extreme cold to Ͼ53°C for extreme heat (reviewed in Refs. 6 and 8). Among them, TRPV1 has received a great deal of attention because it was the first cloned channel that responded to pain-producing heat (Ͼ43°C) and acid stimuli and it is the receptor for capsaicin, the pungent ingredient of hot chili pepper (5, 9). These functional features and the predominant expression of TRPV1 mRNA in the small diameter neurons of the rat dorsal root ganglia (DRG) provide support for TRPV1 as a key player in nociception of primary afferent neurons. Subsequent studies showed that TRPV1 is activated by the endogenous cannabinoid receptor ligand anandamide, N-arachidonyldopamines, and several lipoxygenase products, such as 15-hydroxyeicosatetraenoic acid (10 -13) and its activity is strongly potentiated by inflammatory mediators (14), suggesting a role of TRPV1 besides nociception. Indeed, TRPV1 is expressed in non-sensory tissues (15). Detailed studies hav...

Excitotoxicity has been implicated as the mechanism of neuronal damage resulting from acute insults such as stroke, epilepsy, and trauma, as well as during the progression of adult-onset neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis (ALS). Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. One potential approach to protect against excitotoxic neuronal damage is enhanced glutamate reuptake. The glial glutamate transporter EAAT2 is the quantitatively dominant glutamate transporter and plays a major role in clearance of glutamate. Expression of EAAT2 protein is highly regulated at the translational level. In an effort to identify compounds that can induce translation of EAAT2 transcripts, a cell-based enzyme-linked immunosorbent assay was developed using a primary astrocyte line stably transfected with a vector designed to identify modulators of EAAT2 translation. This assay was optimized for high-throughput screening, and a library of approximately 140,000 compounds was tested. In the initial screen, 293 compounds were identified as hits. These 293 hits were retested at 3 concentrations, and a total of 61 compounds showed a dose-dependent increase in EAAT2 protein levels. Selected compounds were tested in full 12-point dose-response experiments in the screening assay to assess potency as well as confirmed by Western blot, immunohistochemistry, and glutamate uptake assays to evaluate the localization and function of the elevated EAAT2 protein. These hits provide excellent starting points for developing therapeutic agents to prevent excitotoxicity. (Journal of Biomolecular Screening 2010:653-662)