title: Activation of the Ano1 (TMEM16A) chloride channel by calcium is not mediated by calmodulin.
abstract: The Ca(2+)-activated Cl channel anoctamin-1 (Ano1; Tmem16A) plays a variety of physiological roles, including epithelial fluid secretion. Ano1 is activated by increases in intracellular Ca(2+), but there is uncertainty whether Ca(2+) binds directly to Ano1 or whether phosphorylation or additional Ca(2+)-binding subunits like calmodulin (CaM) are required. Here we show that CaM is not necessary for activation of Ano1 by Ca(2+) for the following reasons. (a) Exogenous CaM has no effect on Ano1 currents in inside-out excised patches. (b) Overexpression of Ca(2+)-insensitive mutants of CaM have no effect on Ano1 currents, whereas they eliminate the current mediated by the small-conductance Ca(2+)-activated K(+) (SK2) channel. (c) Ano1 does not coimmunoprecipitate with CaM, whereas SK2 does. Furthermore, Ano1 binds very weakly to CaM in pull-down assays. (d) Ano1 is activated in excised patches by low concentrations of Ba(2+), which does not activate CaM. In addition, we conclude that reversible phosphorylation/dephosphorylation is not required for current activation by Ca(2+) because the current can be repeatedly activated in excised patches in the absence of ATP or other high-energy compounds. Although Ano1 is blocked by the CaM inhibitor trifluoperazine (TFP), we propose that TFP inhibits the channel in a CaM-independent manner because TFP does not inhibit Ano1 when applied to the cytoplasmic side of excised patches. These experiments lead us to conclude that CaM is not required for activation of Ano1 by Ca(2+). Although CaM is not required for channel opening by Ca(2+), work of other investigators suggests that CaM may have effects in modulating the biophysical properties of the channel.
authors.affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322.
authors.forename: Kuai
authors.initials: K
authors.lastname: Yu
authors.forename: Jinqiu
authors.initials: J
authors.lastname: Zhu
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Qu
authors.forename: Yuan-Yuan
authors.initials: YY
authors.lastname: Cui
authors.forename: H Criss
authors.initials: HC
authors.lastname: Hartzell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000075369:Anoctamin-1; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D065101:Chloride Channel Agonists; D018118:Chloride Channels / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data
chemical_list: C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D002147:Calmodulin; D065101:Chloride Channel Agonists; D018118:Chloride Channels; D002118:Calcium