Transient receptor potential vanilloid 1 (TRPV1) is an ion channel that is gated by noxious heat, capsaicin and other diverse stimuli. It is a nonselective cation channel that prefers Ca2+ over Na+. These permeability characteristics, as in most channels, are widely presumed to be static. On the contrary, we found that activation of native or recombinant rat TRPV1 leads to time- and agonist concentration-dependent increases in relative permeability to large cations and changes in Ca2+ permeability. Using the substituted cysteine accessibility method, we saw that these changes were attributable to alterations in the TRPV1 selectivity filter. TRPV1 agonists showed different capabilities for evoking ionic selectivity changes. Furthermore, protein kinase C-dependent phosphorylation of Ser800 in the TRPV1 C terminus potentiated agonist-evoked ionic selectivity changes. Thus, the qualitative signaling properties of TRPV1 are dynamically modulated during channel activation, a process that probably shapes TRPV1 participation in pain, cytotoxicity and neurotransmitter release.

Six of the mammalian transient receptor potential (TRP) ion channel subtypes are nonselective cation channels that can be activated by increases or decreases in ambient temperature. Five of them can alternatively be activated by nonthermal stimuli such as capsaicin [transient receptor potential vanilloid 1 (TRPV1)] or hypo-osmolarity (TRPV2 and TRPV4). No nonthermal stimuli have yet been described for TRPV3, a warmth-gated ion channel expressed prominently in skin keratinocytes. Here, we demonstrate that 2-aminoethoxydiphenyl borate (2-APB), a compound used to inhibit store-operated Ca 2ϩ channels and IP 3 receptors, produces robust activation of recombinant TRPV3 in human embryonic kidney 293 cells with an EC 50 of 28 M. 2-APB also sensitizes TRPV3 to activation by heat, even at subthreshold concentrations. In inside-out membrane patches from TRPV3-expressing cells, 2-APB increases the open probability of TRPV3. Also, whereas heat alone is capable of activating TRPV3-mediated currents in only a small proportion of primary mouse keratinocytes, 2-APB activates heat-evoked, TRPV3-mediated currents in the majority of these cells. Together, these findings identify 2-APB as the first known chemical activator of TRPV3 and enhance the notion that TRPV3 participates in the detection of heat by keratinocytes.

Recently, a family of temperature-activated ion channels has been identified in mammalian and nonmammalian species that appear to contribute to thermosensation. Two of these proteins, TRPV3 and TRPV4, are ion channels activated by modest increases in ambient temperature. Localization studies have indicated that both proteins, in addition to being expressed in sensory neurons, are also expressed in skin keratinocytes. These and other findings have suggested that keratinocytes might act in concert with sensory neurons to perceive our thermal environment. In this study, we demonstrate that primary keratinocytes isolated from mouse skin exhibit two distinct heat-evoked current responses to mild increases in ambient temperature. The more common of these response types bears considerable similarity to responses mediated by recombinant TRPV4, is absent in mice lacking this ion channel, and is restored upon TRPV4 reintroduction. The second, rarer response strongly resembles those mediated by recombinant TRPV3. Together, these findings demonstrate that keratinocytes can indeed act as thermosensory cells and that they do so via at least two distinct transduction mechanisms.The skin forms an interface with our surroundings that is simultaneously protective and perceptual. The outermost skin layer, the epidermis, consists predominantly of stratified epithelial cells that differentiate to form a superficial layer of keratin-filled squamous cells together with dendritic cells, melanocytes, and sensory nerve endings. According to conventional dogma, nerve endings mediate the sensory functions of skin, whereas keratinocytes account for the mechanical and chemical barrier properties of the skin. However, this dichotomy may be overly simplistic. For instance, keratinocytes release substances such as neurotrophins and opioids that can sensitize or desensitize adjacent nerve endings (1, 2). Along similar lines, recent data, described in the next paragraph, have led to speculation that keratinocytes might act as thermosensory cells that transmit information regarding ambient temperature to "warmth-sensitive" afferents.TRPV1 is a cation channel originally identified as the receptor for the pungent vanilloid compound, capsaicin (3). TRPV1 can alternatively be activated by low pH or painfully hot temperatures (i.e. Ͼ42°C). Responses to noxious heat are diminished in mice lacking TRPV1, and responses to moderately hot temperatures (42-52°C) are undetectable in TRPV1 null sensory neurons in vitro. Still, considerable heat responsiveness remains in TRPV1 knockout mice and in skin-nerve preparations explanted from them. These results demonstrate the existence of TRPV1-independent mechanisms of noxious heat transduction and suggest that such mechanisms may involve other cell types in addition to sensory neurons. The process by which mammals detect innocuous warm temperatures is even less well understood than that underlying thermal nociception despite the fact that nerve fibers responsive to warmth (i.e. 30 -42°C) were first identified deca...

Mammalian survival requires constant monitoring of environmental and body temperature. Recently, several members of the transient receptor potential vanilloid (TRPV) subfamily of ion channels have been identified that can be gated by increases in temperature into the warm (TRPV3 and TRPV4) or painfully hot (TRPV1 and TRPV2) range. In rodents, TRPV3 and TRPV4 proteins have not been detected in sensory neurons but are highly expressed in skin epidermal keratinocytes. Here, we show that in response to warm temperatures (>32°C), the mouse 308 keratinocyte cell line exhibits nonselective transmembrane cationic currents and Ca 2؉ influx. Both TRPV3 and TRPV4 are expressed in 308 cells. However, the warmth-evoked responses we observe most closely resemble those mediated by recombinant TRPV4 on the basis of their electrophysiological properties and sensitivity to osmolarity and the phorbol ester, 4␣-phorbol-12,13-didecanoate. Together, these data support the notion that keratinocytes are capable of detecting modest temperature elevations, strongly suggest that TRPV4 participates in these responses, and define a system for the cell biological analysis of warmth transduction.The perception of ambient temperature is a physiological process critical to the maintenance of body temperature and the avoidance of painful or dangerous thermal extremes. Since the identification of the heat-sensing ion channel TRPV1 (1), 1 which is activated by temperatures above 42°C, there has been a significant focus on potential thermosensory functions for this and other ion channels of the transient receptor potential family. TRPV2 (2) and TRPV3 (3-5) were first described as heat transducers operative at very hot (Ͼ52°C) and moderately warm (Ͼ34°C) temperatures, respectively. TRPV4, which was originally identified as an osmosensory ion channel (6 -9), can also be activated by warm temperatures (Ͼ34°C) (10, 11). In addition, two TRP proteins outside of the TRPV subfamily, TRPM8 (12, 13) and ANKTM1 (ankyrin repeat/transmembrane-containing ion channel) (14), have been identified as cold-activated ion channels expressed in sensory neurons.In mammals, the skin is extremely important for the transduction of thermal information and its transmission to the central nervous system. Cutaneous thermosensation has been largely attributed to the sensory nerves that innervate the dermal and epidermal layers of the skin. Recent reports, however, have suggested the possibility that other skin components, most notably keratinocytes, might also participate in temperature sensation. TRPV1 and TRPV2 are highly expressed in distinct subsets of sensory neurons (2). In humans, TRPV3 is also expressed in sensory neurons (5). In contrast, attempts to detect TRPV4 (9, 10) and TRPV3 (3) at the protein level in rodent sensory neurons have been unsuccessful. Rather, immunohistochemical studies of mouse and rat skin have revealed that keratinocytes exhibit the greatest degree of cutaneous TRPV3 (3) and TRPV4 (10) expression. In addition, although peripheral sensory neurons ...

The ability to sense changes in the environment is essential for survival because it permits responses such as withdrawal from noxious stimuli and regulation of body temperature. Keratinocytes, which occupy much of the skin epidermis, are situated at the interface between the external environment and the body's internal milieu, and have long been appreciated for their barrier function against external insults. The recent discovery of temperature-sensitive transient receptor potential vanilloid (TRPV) ion channels in keratinocytes has raised the possibility that these cells also actively participate in acute temperature and pain sensation. To address this notion, we generated and characterized transgenic mice that overexpress TRPV3 in epidermal keratinocytes under the control of the keratin 14 promoter. Compared with wild-type controls, keratinocytes overexpressing TRPV3 exhibited larger currents as well as augmented prostaglandin E 2 (PGE 2 ) release in response to two TRPV3 agonists, 2-aminoethoxydiphenyl borate (2APB) and heat. Thermal selection behavior and heat-evoked withdrawal behavior of naive mice overexpressing TRPV3 were not consistently altered. Upon selective pharmacological inhibition of TRPV1 with JNJ-7203212, however, the keratinocyte-specific TRPV3 transgenic mice showed increased escape responses to noxious heat relative to their wild-type littermates. Coadministration of the cyclooxygenase inhibitor, ibuprofen, with the TRPV1 antagonist decreased inflammatory thermal hyperalgesia in transgenic but not wild-type animals. Our results reveal a previously undescribed mechanism for keratinocyte participation in thermal pain transduction through keratinocyte TRPV3 ion channels and the intercellular messenger PGE 2 .