Association of TRPV1 and TLR4 through the TIR domain potentiates TRPV1 activity by blocking activation-induced desensitization

Abstract

Background We have previously reported that histamine-induced pruritus was attenuated in toll-like receptor 4 (TLR4) knockout mice due to decreased transient receptor potential V1 (TRPV1) sensitivity. Our results implied that TLR4 potentiated TRPV1 activation in sensory neurons; however, the molecular mechanism has yet to be elucidated. In this study, we investigated the molecular mechanisms of TLR4-mediated TRPV1 potentiation using TLR4-deficient sensory neurons and a heterologous expression system. Methods Primary sensory neurons were obtained from wild-type or TLR4 knockout mice, and HEK293T cells expressing TRPV1 and TLR4 were prepared by transient transfection. TRPV1 activity was analyzed by calcium imaging, fluorophotometry, and patch-clamp recording. Subcellular protein distribution was tested by immunocytochemistry and cell surface biotinylation assay. Protein interaction was assessed by western blot and immunoprecipitation assay. Results Direct association between TRPV1 and TLR4 was detected in HEK293T cells upon heterologous TRPV1 and TLR4 expression. In an immunoprecipitation assay using TLR4-deletion mutants and soluble toll/interleukin-1 receptor (TIR) protein, the cytoplasmic TIR domain of TLR4 was required for TLR4-TRPV1 association and TRPV1 potentiation. In TLR4-deficient sensory neurons, the activation-induced desensitization of TRPV1 increased, accompanied by enhanced TRPV1 clearance from the cell membrane upon activation compared to wild-type neurons. In addition, heterologous TLR4 expression inhibited activation-induced TRPV1 endocytosis and lysosomal degradation in HEK293T cells. Conclusion Our data show that direct association between TRPV1 and TLR4 through the TIR domain enhances TRPV1 activity by blocking activation-induced TRPV1 desensitization.