Background and PurposeTMEM16A, also known as anoctamin 1 channel, is a member of the Ca2+-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 ApproachWe 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 Ca2+ imaging and patch-clamp recordings. We also used site-directed mutagenesis to determine the molecular basis of Eact activation of TRPV1. Key ResultsAdministration 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 Ca2+ 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 ImplicationsOur results suggest that Eact activates primary sensory nociceptors and produces both pain and itch responses mainly through direct activation of TRPV1 channels.
[1]Washington Univ, Sch Med, Dept Anesthesiol, Ctr Study Itch, St Louis, MO 63110 USA.
[2]Fudan Univ, Dept Integrat Med & Neurobiol, State Key Lab Med Neurobiol, Shanghai Med Coll, Shanghai 200032, Peoples R China.
[3]Washington Univ, Sch Med, Dept Internal Med, St Louis, MO 63110 USA.
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