title: Eact, a small molecule activator of TMEM16A, activates TRPV1 and elicits pain- and itch-related behaviours.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  Our results suggest that Eact activates primary sensory nociceptors and produces both pain and itch responses mainly through direct activation of TRPV1 channels.
authors.affiliation: Department of Anesthesiology, The Center for the Study of Itch, Washington University School of Medicine, St. Louis, MO, 63110, USA.|Department of Integrative Medicine and Neurobiology, State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
authors.forename: Shenbin
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Anesthesiology, The Center for the Study of Itch, Washington University School of Medicine, St. Louis, MO, 63110, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Feng
authors.affiliation: Department of Anesthesiology, The Center for the Study of Itch, Washington University School of Medicine, St. Louis, MO, 63110, USA.
authors.forename: Jialie
authors.initials: J
authors.lastname: Luo
authors.affiliation: Department of Anesthesiology, The Center for the Study of Itch, Washington University School of Medicine, St. Louis, MO, 63110, USA.
authors.forename: Pu
authors.initials: P
authors.lastname: Yang
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Brett
authors.affiliation: Department of Anesthesiology, The Center for the Study of Itch, Washington University School of Medicine, St. Louis, MO, 63110, USA.
authors.forename: Hongzhen
authors.initials: H
authors.lastname: Hu
mesh_terms: D000818:Animals; D000075369:Anoctamin-1; D001522:Behavior, Animal / Q000187:drug effects*; D001549:Benzamides; D002478:Cells, Cultured; D065101:Chloride Channel Agonists / Q000494:pharmacology*; D018118:Chloride Channels / Q000378:metabolism*; D005727:Ganglia, Spinal / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D020297:Mice, Congenic; D009619:Nociceptors / Q000187:drug effects / Q000378:metabolism; D010146:Pain / Q000378:metabolism* / Q000523:psychology; D011537:Pruritus / Q000378:metabolism* / Q000523:psychology; D051381:Rats; D050916:TRPV Cation Channels / Q000819:agonists* / Q000235:genetics; D013844:Thiazoles
chemical_list: C000612090:(3,4,5-trimethoxy-N-(2-methoxyethyl)-N-(4-phenyl-2-thiazolyl)benzamide; C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D001549:Benzamides; D065101:Chloride Channel Agonists; D018118:Chloride Channels; D050916:TRPV Cation Channels; C493921:TRPV1 protein, mouse; D013844:Thiazoles