The pharmacological desensitization of receptors is a fundamental mechanism for regulating the activity of neuronal systems. The TRPA1 channel plays a key role in the processing of noxious information and can undergo functional desensitization by unknown mechanisms. Here we show that TRPA1 is desensitized by homologous (mustard oil; a TRPA1 agonist) and heterologous (capsaicin; a TRPV1 agonist) agonists via Ca 2+ -independent and Ca 2+ -dependent pathways, respectively, in sensory neurons. The pharmacological desensitization of TRPA1 by capsaicin and mustard oil is not influenced by activation of protein phosphatase 2B. However, it is regulated by phosphatidylinositol-4,5-bisphosphate depletion after capsaicin, but not mustard oil, application. Using a biosensor, we establish that capsaicin, unlike mustard oil, consistently activates phospholipase C in sensory neurons. We next demonstrate that TRPA1 desensitization is regulated by TRPV1, and it appears that mustard oil-induced TRPA1 internalization is prevented by coexpression with TRPV1 in a heterologous expression system and in sensory neurons. In conclusion, we propose novel mechanisms whereby TRPA1 activity undergoes pharmacological desensitization through multiple cellular pathways that are agonist dependent and modulated by TRPV1.

Several lines of evidence suggest that TRPA1 and TRPV1 mutually control the transduction of inflammation-induced noxious stimuli in sensory neurons. It was recently shown that certain TRPA1 properties are modulated by TRPV1. However, direct interaction between TRPA1 and TRPV1 as well as regulation of TRPA1 intrinsic characteristics by the TRPV1 channel have not been examined. To address these questions, we have studied a complex formation between TRPA1 and TRPV1 and characterized the influence of TRPV1 on single channel TRPA1-mediated currents. Co-immunoprecipitation analysis revealed direct interactions between TRPA1 and TRPV1 in an expression system as well as in sensory neurons. Data generated with total internal reflection fluorescence-based fluorescence resonance energy transfer indicate that a TRPA1-TRPV1 complex can be formed on the plasma membrane. . In summary, our results support the hypothesis that TRPV1 and TRPA1 form a complex and that TRPV1 influences intrinsic characteristics of the TRPA1 channel.Studies using TRPV1-and TRPA1-null mutant mouse lines (1-3), TRPA1 antisense knockdown (4), and in vivo effects of TRPA1 and TRPV1 antagonists (5, 6) have demonstrated that TRPV1 and TRPA1 channels play an important role in the development of hyperalgesia in certain inflammatory and neuropathic pain models.Like most mammalian TRP channels, TRPV1 and possibly TRPA1 function as homotetramers (7,8). In addition, TRP channels belonging to the same subfamilies (i.e. the V, C, M, or P subfamilies) can interact and form heteromers (7). It has also been noted that most heteromeric TRP channel complexes appear to consist of subunit combinations only within relatively narrow confines of phylogenetic subfamilies (7). Recent reports indicate that the native TRPA1 channel has characteristics different from those of homomeric TRPA1 expressed in cell lines (9, 10). Interestingly, these characteristics of TRPA1 are restored similarly to characteristics recorded from native cells when TRPA1 and TRPV1 are co-expressed in sensory neurons or expression systems (9). Further, there is evidence suggesting that these channels could heterologously interregulate each other's activities and characteristics. Thus, pharmacological cross-desensitization between capsaicin and mustard oil responses was noted and characterized (10 -13). Transmission of inflammatory stimuli by nociceptors (i.e. damage-sensing sensory neurons) is also mutually controlled by TRPA1 and TRPV1 channels (1, 3). It was suggested that this functional interaction between TRPV1 and TRPA1 could occur either indirectly via recruitment of second messengers, such as intracellular Ca 2ϩ ([Ca 2ϩ ] i ) (1, 14), or directly, involving interaction of these channels within a complex (9,15).Given previously published results, the present study assesses the following questions: (i) whether TRPA1 and TRPV1 channels could assemble into a complex on the plasma membrane; (ii) whether interactions of TRPA1 with TRPV1 affect intrinsic (i.e. single channel) characteristics of TRPA1...

Mood disorders cause much suffering and are the single greatest cause of lost productivity worldwide. Although multiple medications, along with behavioral therapies, have proven effective for some individuals, millions of people lack an effective therapeutic option. A common serotonin (5-HT) transporter (5-HTT/SERT, SLC6A4) polymorphism is believed to confer lower 5-HTT expression in vivo and elevates risk for multiple mood disorders including anxiety, alcoholism, and major depression. Importantly, this variant is also associated with reduced responsiveness to selective 5-HT reuptake inhibitor antidepressants. We hypothesized that a reduced antidepressant response in individuals with a constitutive reduction in 5-HTT expression could arise because of the compensatory expression of other genes that inactivate 5-HT in the brain. A functionally upregulated alternate transporter for 5-HT may prevent extracellular 5-HT from rising to levels sufficiently high enough to trigger the adaptive neurochemical events necessary for therapeutic benefit. Here we demonstrate that expression of the organic cation transporter type 3 (OCT3, SLC22A3), which also transports 5-HT, is upregulated in the brains of mice with constitutively reduced 5-HTT expression. Moreover, the OCT blocker decynium-22 diminishes 5-HT clearance and exerts antidepressantlike effects in these mice but not in WT animals. OCT3 may be an important transporter mediating serotonergic signaling when 5-HTT expression or function is compromised.5HTTLPR ͉ antidepressant ͉ polymorphism ͉ hippocampus ͉ chronamperometry

SUMMARY TRPA1 and TRPV1 are crucial pain mediators, but how their interaction contributes to persistent pain is unknown. Here, we identify Tmem100 as a potentiating modulator of TRPA1-V1 complexes. Tmem100 is co-expressed and forms a complex with TRPA1 and TRPV1 in DRG neurons. Tmem100-deficient mice show a reduction in inflammatory mechanical hyperalgesia and TRPA1- but not TRPV1-mediated pain. Single-channel recording in a heterologous system reveals that Tmem100 selectively potentiates TRPA1 activity in a TRPV1-dependent manner. Mechanistically, Tmem100 weakens the association of TRPA1 and TRPV1, thereby releasing the inhibition of TRPA1 by TRPV1. A Tmem100 mutant, Tmem100-3Q, exerts the opposite effect, i.e., it enhances the association of TRPA1 and TRPV1 and strongly inhibits TRPA1. Strikingly, a cell-permeable peptide (CPP) containing the C-terminal sequence of Tmem100-3Q mimics its effect and inhibits persistent pain. Our study unveils a context-dependent modulation of the TRPA1-V1 complex, and Tmem100-3Q CPP is a promising pain therapy.

Certain phosphorylation events are tightly controlled by scaffolding proteins such as A-kinase anchoring protein (AKAP). On nociceptive terminals, phosphorylation of transient receptor potential channel type 1 (TRPV1) results in the sensitization to many different stimuli, contributing to the development of hyperalgesia. In this study, we investigated the functional involvement of AKAP150 in mediating sensitization of TRPV1, and found that AKAP150 is co-expressed in trigeminal ganglia (TG) neurons from rat and associates with TRPV1. Furthermore, siRNA-mediated knock-down of AKAP150 expression led to a significant reduction in PKA phosphorylation of TRPV1 in cultured TG neurons. In CHO cells, the PKA RII binding site on AKAP was necessary for PKA enhancement of TRPV1-mediated Ca2+-accumulation. In addition, AKAP150 knock-down in cultured TG neurons attenuated PKA sensitization of TRPV1 activity and in vivo administration of an AKAP antagonist significantly reduced prostaglandin E2 sensitization to thermal stimuli. These data suggest that AKAP150 functionally regulates PKA-mediated phosphorylation/sensitization of the TRPV1 receptor.

Peripheral opioid analgesia is increased substantially after inflammation. We evaluated the hypothesis that an inflammatory mediator, bradykinin (BK), evokes functional competence of the ␦-opioid receptor (DOR) for inhibiting trigeminal ganglia (TG) sensory neurons. We also evaluated whether BK evokes trafficking of the DOR to the plasma membrane. Rat TG cultures were pretreated with BK (

Sex dependency in pain perception is well documented and is thought to be attributable to the effect of reproductive hormones on nociceptive processing. In the present study, we evaluated whether estradiol alters gene transcription in the trigeminal ganglia (TG) of ovariectomized rats (OVX). These experiments demonstrated a dramatic (40-fold) upregulation of prolactin (PRL) expression in TG by 17-␤-estradiol (E2). PRL expression was restricted to TG neurons and was highly overlapped with transient potential receptor vanilloid type 1 (TRPV1) (ϳ90%) in TG. Additionally, PRL is released from neurons during stimulation. Both forms of PRL receptors (PRLRs), short and long, were also present in TG neurons. Moreover, expression of the long PRLRs was under control of estradiol. We next evaluated the novel hypothesis that PRL acts as a neuromodulator of sensory neurons. PRL pretreatment significantly enhanced capsaicin-evoked inward currents, calcium influx, and immunoreactive calcitonin gene-related peptide release from cultured TG neurons. This PRL modulation of capsaicin responses was abolished by withdrawal of E2 from TG cultures. Biochemical analysis demonstrated that PRL increased (Ͼ50%) phosphorylation levels of TRPV1 in TG. In a behavioral test, PRL pretreatment significantly potentiated capsaicin-evoked nocifensive behavior in female rats at proestrous and in OVX rats after E2 treatment. The in vivo potentiating effect of PRL on capsaicin responses was also dependent on E2. Collectively, these data demonstrate that PRL is a novel modulator of sensory neurons tightly regulated by E2. These findings are consistent with the hypothesis that PRL could contribute to the development of certain pain disorders, possibly including those modulated by estrogen.

Cannabinoids are known to have multiple sites of action in the nociceptive system, leading to reduced pain sensation. However, the peripheral mechanism(s) by which this phenomenon occurs remains an issue that has yet to be resolved. Because phosphorylation of TRPV1 (transient receptor potential subtype V1) plays a key role in the induction of thermal hyperalgesia in inflammatory pain models, we evaluated whether the cannabinoid agonist WIN 55,212-2 (WIN) regulates the phosphorylation state of TRPV1. Here, we show that treatment of primary rat trigeminal ganglion cultures with WIN led to dephosphorylation of TRPV1, specifically at threonine residues. Utilizing Chinese hamster ovary cell lines, we demonstrate that Thr 144 and Thr 370 were dephosphorylated, leading to desensitization of the TRPV1 receptor. This post-translational modification occurred through activation of the phosphatase calcineurin (protein phosphatase 2B) following WIN treatment. Furthermore, knockdown of TRPA1 (transient receptor potential subtype A1) expression in sensory neurons by specific small interfering RNA abolished the WIN effect on TRPV1 dephosphorylation, suggesting that WIN acts through TRPA1. We also confirm the importance of TRPA1 in WIN-induced dephosphorylation of TRPV1 in Chinese hamster ovary cells through targeted expression of one or both receptor channels. These results imply that the cannabinoid WIN modulates the sensitivity of sensory neurons to TRPV1 activation by altering receptor phosphorylation. In addition, our data could serve as a useful strategy in determining the potential use of certain cannabinoids as peripheral analgesics.Cannabinoids have been shown to exert anti-inflammatory and anti-hyperalgesic effects via peripheral site(s) of action in several pain models (1-5). These effects are thought to be mediated by cannabinoid type 1 (CB1) 4 and/or 2 (CB2) receptor activation, both peripherally and centrally (4 -7). Cannabinoids could exert their effects by acting on CB1/CB2 receptors located on sensory neurons and/or other peripheral cells influencing sensory neuronal function (8). However, there is a Ͻ5-10% co-localization of metabotropic CB1/CB2 receptors with nociceptive neuronal markers such as TRPV1 (transient receptor potential subtype V1) and calcitonin gene-related peptide in trigeminal and dorsal root ganglion neurons (9 -11), suggesting that cannabinoids could act on nociceptors through non-CB1/CB2 receptor mechanism(s). Certain cannabinoids have been shown to activate channels such as TRPV1, including arachidonyl-2-chloroethylamide (ACEA) (12), N-arachidonoyldopamine (13), and anandamide (14), as well as TRPA1 (transient receptor potential subtype A1), including ⌬ 9 -tetrahydrocannabinol (15). In addition, the synthetic cannabinoid R(ϩ)-WIN 55,212-2 (WIN) has demonstrated non-CB1/CB2 receptor activities in trigeminal ganglia (11). The results from these studies suggest that cannabinoids may activate calcium channel function similar to non-cannabinoid transient receptor potential agonists, including the...