title: IFN-γ-mediated reduction of large-conductance, Ca2+-activated, voltage-dependent K+ (BK) channel activity in airway epithelial cells leads to mucociliary dysfunction.
abstract: Effective mucociliary clearance (MCC) depends in part on adequate airway surface liquid (ASL) volume to maintain an appropriate periciliary fluid height that allows normal ciliary activity. Apically expressed large-conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels provide an electrochemical gradient for Cl(-) secretion and thus play an important role for adequate airway hydration. Here we show that IFN-γ decreases ATP-mediated apical BK activation in normal human airway epithelial cells cultured at the air-liquid interface. IFN-γ decreased mRNA levels of KCNMA1 but did not affect total protein levels. Because IFN-γ upregulates dual oxidase (DUOX)2 and therefore H2O2 production, we hypothesized that BK inactivation could be mediated by BK oxidation. However, DUOX2 knockdown did not affect the IFN-γ effect on BK activity. IFN-γ changed mRNA levels of the BK β-modulatory proteins KCNMB2 (increased) and KCNMB4 (decreased) as well as leucine-rich repeat-containing protein (LRRC)26 (decreased). Mallotoxin, a BK opener only in the absence of LRRC26, showed that BK channels lost their association with LRRC26 after IFN-γ treatment. Finally, IFN-γ caused a decrease in ciliary beating frequency that was immediately rescued by apical fluid addition, suggesting that it was due to ASL volume depletion. These data were confirmed with direct ASL measurements using meniscus scanning. Overexpression of KCNMA1, the pore-forming subunit of BK, overcame the reduction of ASL volume induced by IFN-γ. Key experiments were repeated in cystic fibrosis cells and showed the same results. Therefore, IFN-γ induces mucociliary dysfunction through BK inactivation.
authors.affiliation: 1600 NW 10 Ave., RMSB 7063, Miami, FL 33136,. msalathe@med.miami.edu.
authors.forename: Dahis
authors.initials: D
authors.lastname: Manzanares
authors.forename: Maria
authors.initials: M
authors.lastname: Srinivasan
authors.forename: Samuel T
authors.initials: ST
authors.lastname: Salathe
authors.forename: Pedro
authors.initials: P
authors.lastname: Ivonnet
authors.forename: Nathalie
authors.initials: N
authors.lastname: Baumlin
authors.forename: John S
authors.initials: JS
authors.lastname: Dennis
authors.forename: Gregory E
authors.initials: GE
authors.lastname: Conner
authors.forename: Matthias
authors.initials: M
authors.lastname: Salathe
mesh_terms: D029681:Arabidopsis Proteins / Q000378:metabolism; D001980:Bronchi / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism; D000074623:Dual Oxidases; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019751:Intramolecular Transferases / Q000378:metabolism; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000235:genetics / Q000378:metabolism*; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits / Q000235:genetics / Q000378:metabolism*; D009079:Mucociliary Clearance / Q000187:drug effects / Q000502:physiology*; D019255:NADPH Oxidases / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014132:Trachea / Q000166:cytology / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D002712:Chlorides; C494841:KCNMA1 protein, human; C588418:KCNMB2 protein, human; C410183:KCNMB4 protein, human; C539913:LRRC26 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D007371:Interferon-gamma; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C431515:DUOX2 protein, human; D019751:Intramolecular Transferases; C568454:marneral synthase, Arabidopsis
keywords: BK channel; H2O2; IFN-γ; airway surface liquid; ciliary activity; dual oxidase