The capsaicin receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been reported that ATP, one of the inflammatory mediators, potentiates the VR1 currents evoked by capsaicin or protons and reduces the temperature threshold for activation of VR1 through metabotropic P2Y 1 receptors in a protein Kinase C (PKC)-dependent pathway, suggesting the phosphorylation of VR1 by PKC. In this study, direct phosphorylation of VR1 upon application of phorbol 12-myristate 13-acetate (PMA) was proven biochemically in cells expressing VR1. An in vitro kinase assay using glutathione S-transferase fusion proteins with cytoplasmic segments of VR1 showed that both the first intracellular loop and carboxyl terminus of VR1 were phosphorylated by PKC⑀. Patch clamp analysis of the point mutants where Ser or Thr residues were replaced with Ala in the total 16 putative phosphorylation sites showed that two Ser residues, Ser 502 and Ser 800 were involved in the potentiation of the capsaicinevoked currents by either PMA or ATP. In the cells expressing S502A/S800A double mutant, the temperature threshold for activation was not reduced upon PMA treatment. The two sites would be promising targets for the development of substance modulating VR1 function, thereby reducing pain.The sensation of pain allows us to recognize injury and triggers appropriate protective responses. A specific population of primary afferent neurons called nociceptors are known to be involved in the detection of noxious thermal, mechanical, or chemical stimuli and can be distinguished by their sensitivity to capsaicin, the pungent ingredient in hot chili peppers (1-4). The capsaicin receptor VR1 1 is a nonspecific cation channel with six transmembrane domains expressed predominantly in unmyelinated C fibers and activated not only by capsaicin but also by noxious heat (with a thermal threshold Ͼ 43°C) or protons (acidification), both of which cause pain in vivo (5-9). This sensitivity of VR1 to multiple noxious stimuli might explain certain properties of so called polymodal nociceptors. Furthermore, analyses of mice lacking VR1 have shown that VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia, further suggesting a critical role for VR1 in the detection or modulation of pain (10, 11).Tissue damage associated with infection, inflammation, or ischemia produces an array of chemical mediators that activate or sensitize nociceptor terminals to elicit or exacerbate pain at the site of injury in addition to the release of the mediators from the niciceptor terminals themselves known as neurogenic inflammation. An important component of this pro-algesic response, adenosine 5Ј-triphosphate (ATP), has recently been found to potentiate the VR1 currents evoked by capsaicin or protons through metabotropic P2Y 1 receptor activation in a protein kinase C (PKC)-dependent pathway (12). In the presence of extracellular ATP, the temperature...

The capsaicin receptor, TRPV1 (VR1), is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. Extracellular Ca 2؉ -dependent desensitization of TRPV1 observed in patch-clamp experiments when using both heterologous expression systems and native sensory ganglia is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. Here, we show that the Ca 2؉ -binding protein calmodulin binds to a 35-aa segment in the C terminus of TRPV1, and that disruption of the calmodulin-binding segment prevents TRPV1 desensitization. Compounds that interfere with the 35-aa segment could therefore prove useful in the treatment of pain.

Important mechanisms that regulate inhibitory and facilitatory effects on TRPV1-mediated nociception are desensitization and phosphorylation, respectively. Using Ca2+-imaging, we have previously shown that desensitization of TRPV1 upon successive capsaicin applications was reversed by protein kinase C activation in dorsal root ganglion neurons and CHO cells. Here, using both Ca2+-imaging and patch-clamp methods, we show that PMA-induced activation of PKCepsilon is essential for increased sensitivity of desensitized TRPV1. TRPV1 has two putative substrates S502 and S800 for PKCepsilon-mediated phosphorylation. Patch-clamp analysis showed that contribution of single mutant S502A or S800A towards increased sensitivity of desensitized TRPV1 is indistinguishable from that observed in a double mutant S502A/S800A. Since S502 is a non-specific substrate for TRPV1 phosphorylation by kinases like PKC, PKA or CAMKII, evidence for a role of PKC specific substrate S800 was investigated. Evidence for in vivo phosphorylation of TRPV1 at S800 was demonstrated for the first time. We also show that the expression level of PKCepsilon paralleled the amount of phosphorylated TRPV1 protein using an antibody specific for phosphorylated TRPV1 at S800. Furthermore, the anti-phosphoTRPV1 antibody detected phosphorylation of TRPV1 in mouse and rat DRG neurons and may be useful for research regarding nociception in native tissues. This study, therefore, identifies PKCepsilon and S800 as important therapeutic targets that may help regulate inhibitory effects on TRPV1 and hence its desensitization.

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Cell movement is driven by the coordinated regulation of cytoskeletal reorganization through Rho GTPases downstream of integrin and growth-factor receptor signaling. We have reported that mDia, a target protein of Rho, interacts with Src and DIP. Here we show that DIP binds to p190RhoGAP and Vav2, and that DIP is phosphorylated by Src and mediates the phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation. When endogenous DIP was inhibited by expressing dominant-negative mutants of DIP or siRNA, phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation was diminished, and EGF-induced actin organization, distribution of p190RhoGAP and Vav2, and cell movement were affected. Therefore, DIP seems to transfer the complex of the three proteins from cytosol to beneath the membrane, and the three proteins, in turn, can be phosphorylated by Src. DIP inactivated Rho and activated Rac following EGF stimulation in the membrane fraction. Thus, DIP acts as a regulatory molecule causing Src kinase-dependent feedback modulation of Rho GTPases downstream of Rho-mDia upon EGF stimulation, and plays an important role in cell motility.

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