Eact, a small molecule activator of TMEM16A, activates TRPV1 and elicits pain‐ and itch‐related behaviours

Background and Purpose TMEM16A, also known as anoctamin 1 channel, is a member of the Ca 2 + ‐activated chloride channels family and serves as a heat sensor in the primary nociceptors. Eact is a recently discovered small molecule activator of the TMEM16A channel. Here, we asked if Eact produces pain‐ and itch‐related responses in vivo and investigated the cellular and molecular basis of Eact‐elicited responses in dorsal root ganglia (DRG) neurons. Experimental Approach We employed behavioural testing combined with pharmacological inhibition and genetic ablation approaches to identify transient receptor potential vanilloid 1 (TRPV1) as the prominent mediator for Eact‐evoked itch‐ or pain‐related responses. We investigated the effects of Eact on TRPV1 and TMEM16A channels expressed in HEK293T cells and in DRG neurons isolated from wild type and Trpv1 − / − mice using Ca 2 + imaging and patch‐clamp recordings. We also used site‐directed mutagenesis to determine the molecular basis of Eact activation of TRPV1. Key Results Administration of Eact elicited both itch‐ and pain‐related behaviours. Unexpectedly, the Eact‐elicited behavioural responses were dependent on the function of TRPV1, as shown by pharmacological inhibition and genetic ablation studies. Eact activated membrane currents and increased intracellular free Ca 2 + in both TRPV1‐expressing HEK293T cells and isolated DRG neurons in a TRPV1‐dependent manner. Eact activation of the TRPV1 channel was severely attenuated by mutations disrupting the capsaicin‐binding sites. Conclusions and Implications Our results suggest that Eact activates primary sensory nociceptors and produces both pain and itch responses mainly through direct activation of TRPV1 channels.