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. The fluorescence resonance energy transfer interaction between TRPA1 and TRPV1 channels is as effective as for TRPV1 or TRPA1 homomers. Single channel analysis in a heterologous expression system and in sensory neurons of wild type and TRPV1 knock-out mice demonstrated that co-expression of TRPV1 with TRPA1 results in outward rectification of single channel mustard oil (I(MO)) current-voltage relationships (I-V) and substantial modulation of the open probability at negative holding potentials. TRPV1 also does not influence the characteristics of single channel I(MO) in Ca(2+)-free extracellular solution. However, association of TRPA1 with TRPV1 was not affected in Ca(2+)-free media. To assess a role of intracellular Ca(2+) in TRPV1-dependent modulation of TRPA1 modulation, the TRPA1-mediated single channel WIN55,212-2-gated current (I(WIN)) was recorded in inside-out configuration. Our data indicate that single channel properties of TRPA1 are regulated by TRPV1 independently of intracellular Ca(2+). In summary, our results support the hypothesis that TRPV1 and TRPA1 form a complex and that TRPV1 influences intrinsic characteristics of the TRPA1 channel.

Objective: Transient receptor potential ankyrin-1 (TRPA1) and transient receptor potential vanilloid-1 (TRPV1) are calcium (Ca²⁺)-permeable ion channels mostly known as pain receptors in sensory neurons. However, growing evidence suggests their crucial involvement in the pathogenesis of IBD. We explored the possible contribution of TRPA1 and TRPV1 to T-cell-mediated colitis. Design: We evaluated the role of Trpa1 gene deletion in two models of experimental colitis (ie, interleukin-10 knockout and T-cell-adoptive transfer models). We performed electrophysiological and Ca²⁺ imaging studies to analyse TRPA1 and TRPV1 functions in CD4+ T cells. We used genetic and pharmacological approaches to evaluate TRPV1 contribution to the phenotype of Trpa1^−/− CD4+ T cells. We also analysed TRPA1 and TRPV1 gene expression and TRPA1⁺TRPV1⁺ T cell infiltration in colonic biopsies from patients with IBD. Results: We identified a protective role for TRPA1 in T-cell-mediated colitis. We demonstrated the functional expression of TRPA1 on the plasma membrane of CD4+ T cells and identified that Trpa1^−/− CD4+ T cells have increased T-cell receptor-induced Ca²⁺ influx, activation profile and differentiation into Th1-effector cells. This phenotype was abrogated upon genetic deletion or pharmacological inhibition of the TRPV1 channel in mouse and human CD4+ T cells. Finally, we found differential regulation of TRPA1 and TRPV1 gene expression as well as increased infiltration of TRPA1⁺TRPV1⁺ T cells in the colon of patients with IBD. Conclusions: Our study indicates that TRPA1 inhibits TRPV1 channel activity in CD4+ T cells, and consequently restrains CD4+ T-cell activation and colitogenic responses. These findings may therefore have therapeutic implications for human IBD.

Although the cannabinoid agonistsR-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)-(1-naphthalenyl) methanone mesylate [WIN 55,212-2 (WIN)] and (R,S)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) exert peripheral antihyperalgesia in inflammatory pain models, the mechanism for cannabinoid-induced inhibition of nociceptive sensory neurons has not been fully studied. Because TRPV1 and TRPA1 channels play important roles in controlling hyperalgesia in inflammatory pain models, we investigated their modulation by WIN and AM1241. The applications of WIN (>5 μm) and AM1241 (>30 μm) inhibit responses of sensory neurons to capsaicin and mustard oil. To determine potential mechanisms for the inhibition, we evaluated cannabinoid effects on nociceptors. WIN and AM1241 excite sensory neurons in a concentration-dependent manner via a nonselective Ca²⁺-permeable channel. The expression of TRP channels in CHO cells demonstrates that both WIN and AM1241 activate TRPA1 and, by doing so, attenuate capsaicin and mustard oil responses. Using TRPA1-specific small interfering RNA or TRPA1-deficient mice, we show that the TRPA1 channel is a sole target through which WIN and mustard oil activate sensory neurons. In contrast, AM1241 activation of sensory neurons is mediated by TRPA1 and an unknown channel. The knockdown of TRPA1 activity in neurons completely eliminates the desensitizing effects of WIN and AM1241 on capsaicin-activated currents. Furthermore, the WIN- or AM1241-induced inhibition of capsaicin-evoked nocifensive behavior via peripheral actions is reversed in TRPA1 null-mutant mice. Together, this study demonstrates that certain cannabinoids exert their peripheral antinocifensive actions via activation of the TRPA1 channel on sensory neurons.