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E-mail Address : kyu3@emory.edu

Author contributions: K. Yu conceived, designed, and performed experiments, analyzed data, prepared figures, and wrote the manuscript.

J. Zhu designed and performed experiments, analyzed data, and prepared figures. Z. Qu designed and performed experiments and analyzed data.

Y.-Y. Cui designed and performed experiments and analyzed data. H.C. Hartzell conceived and designed experiments, analyzed data, and wrote the manuscript.

Angus C. Nairn served as editor.

We thank Drs. Min Goo Lee and Jinsei Jung (Yonsei University, Seoul, South Korea) for the GST-CaM and human Ano1(ac) constructs and advice on the pull-down assay, John Adelman for the SK2 and CaM234 and CaM1234 cDNA clones, Rob West for the DOG1.1 antibody, and Uhtaek Oh for the mAno1(ac) clone.

The authors have no conflicting financial interests.

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Research Funding:

This work is supported by grants from the National Institutes of Health (GM60448 and EY014852) and a pilot grant from the Center for Cystic Fibrosis Research, Children’s Healthcare of Atlanta and Emory University School of Medicine.

Activation of the Ano1 (TMEM16A) chloride channel by calcium is not mediated by calmodulin

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Journal Title:

Journal of General Physiology

Volume:

Volume 143, Number 2

Publisher:

, Pages 253-267

Type of Work:

Article | Final Publisher PDF

Abstract:

The Ca2+-activated Cl channel anoctamin-1 (Ano1; Tmem16A) plays a variety of physiological roles, including epithelial fluid secretion. Ano1 is activated by increases in intracellular Ca2+, but there is uncertainty whether Ca2+ binds directly to Ano1 or whether phosphorylation or additional Ca2+-binding subunits like calmodulin (CaM) are required. Here we show that CaM is not necessary for activation of Ano1 by Ca2+ for the following reasons. (a) Exogenous CaM has no effect on Ano1 currents in inside-out excised patches. (b) Overexpression of Ca2+-insensitive mutants of CaM have no effect on Ano1 currents, whereas they eliminate the current mediated by the small-conductance Ca2+-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 Ba2+, which does not activate CaM. In addition, we conclude that reversible phosphorylation/dephosphorylation is not required for current activation by Ca2+ 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 Ca2+. Although CaM is not required for channel opening by Ca2+, work of other investigators suggests that CaM may have effects in modulating the biophysical properties of the channel.

Copyright information:

© 2014 Yu et al.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).

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