title: Protons inhibit anoctamin 1 by competing with calcium.
abstract: Cl(-) efflux through Ca(2+)-activated Cl(-) channels (CaCCs) in secretory epithelial cells plays a key role in the regulation of fluid secretion. The fluid and electrolyte secretion is closely related to intracellular pH. CaCCs have been known to be inhibited by intracellular acid. However, the molecular mechanism for the inhibition remains unknown. Anoctamin 1 (ANO1) is a Ca(2+)-activated Cl(-) channel that mediates numerous physiological functions including fluid secretion in secretory epithelia. However, little is known about whether ANO1 can be modulated by change of intracellular pH. Here, we demonstrate that Ca(2+)-induced activation of ANO1 and its homolog ANO2 are strongly inhibited by intracellular acid. Intracellular acid caused a rightward shift of the concentration-response curve of Ca(2+) in activating ANO1 and ANO2. To identify the location of the acid-induced inhibition, mutations were made on each of all histidine residues in cytoplasmic part of ANO1. However, none of the His-mutant showed the reduction in the acid-induced inhibition. Furthermore, mutation on Glu- or Asp-residues in the multiple acidic-amino acid regions was ineffective in blocking the acid-induced inhibition. Because the Ca(2+)-binding site of a fungal anoctamin (nhTMEM16) was uncovered by crystallography, mutagenesis was performed in this region. Surprisingly, mutations at Glu, Asp or Asn residues in the hydrophobic core that are known to be essential for Ca(2+)-induced activation of ANO1 blocked the acid-induced inhibition. These results suggest that protons interfere with Ca(2+) at the Ca(2+) binding site of ANO1. These findings provide a molecular mechanism underlying the acid-induced inhibition of ANO1, which may contribute to control fluid and electrolyte secretion in the secretory epithelia.
authors.affiliation: Sensory Research Center, Creative Research Initiatives, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Hyeyeon
authors.initials: H
authors.lastname: Chun
authors.affiliation: Sensory Research Center, Creative Research Initiatives, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Hawon
authors.initials: H
authors.lastname: Cho
authors.affiliation: Sensory Research Center, Creative Research Initiatives, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Jimi
authors.initials: J
authors.lastname: Choi
authors.affiliation: Sensory Research Center, Creative Research Initiatives, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Jesun
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Physical Education, College of Art and Physical Education, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Sung Min
authors.initials: SM
authors.lastname: Kim
authors.affiliation: Sensory Research Center, Creative Research Initiatives, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Hyungsup
authors.initials: H
authors.lastname: Kim
authors.affiliation: Sensory Research Center, Creative Research Initiatives, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea; Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 151-742, Republic of Korea. Electronic address: utoh@snu.ac.kr.
authors.forename: Uhtaek
authors.initials: U
authors.lastname: Oh
mesh_terms: D000075369:Anoctamin-1; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011522:Protons
chemical_list: C476213:ANO1 protein, human; D000075369:Anoctamin-1; D018118:Chloride Channels; D009363:Neoplasm Proteins; D011522:Protons; D002118:Calcium
keywords: ANO2; Acid; Anoctamin 1 (ANO1); Calcium; E(act); Secretion