title: The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit.
abstract: Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the β and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.
authors.affiliation: From the Vascular Biology Center, rlucas@augusta.edu.|the Department of Pharmacology and Toxicology, and.|the Division of Pulmonary and Critical Care Medicine, Medical College of Georgia, Augusta, Georgia 30912.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Lucas
authors.affiliation: the Department of Physiology, Emory University, Atlanta, Georgia 30322.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yue
authors.affiliation: the Department of Physiology, Emory University, Atlanta, Georgia 30322.|the Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida 32610.
authors.forename: Abdel
authors.initials: A
authors.lastname: Alli
authors.affiliation: the Department of Physiology, Emory University, Atlanta, Georgia 30322.
authors.forename: Billie Jeanne
authors.initials: BJ
authors.lastname: Duke
authors.affiliation: the Department of Physiology, Emory University, Atlanta, Georgia 30322.
authors.forename: Otor
authors.initials: O
authors.lastname: Al-Khalili
authors.affiliation: the Department of Physiology, Emory University, Atlanta, Georgia 30322.
authors.forename: Tiffany L
authors.initials: TL
authors.lastname: Thai
authors.affiliation: the Department of Pulmonology, Saarland University, D-66421 Homburg, Germany.
authors.forename: Jürg
authors.initials: J
authors.lastname: Hamacher
authors.affiliation: From the Vascular Biology Center.
authors.forename: Supriya
authors.initials: S
authors.lastname: Sridhar
authors.affiliation: the Department of Chemistry, Augusta University, Augusta, Georgia 30912.
authors.forename: Iryna
authors.initials: I
authors.lastname: Lebedyeva
authors.affiliation: From the Vascular Biology Center.
authors.forename: Huabo
authors.initials: H
authors.lastname: Su
authors.affiliation: Apeptico Research and Development, 1150 Vienna, Austria.
authors.forename: Susan
authors.initials: S
authors.lastname: Tzotzos
authors.affiliation: Apeptico Research and Development, 1150 Vienna, Austria.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Fischer
authors.affiliation: the Department of Chemistry, Augusta University, Augusta, Georgia 30912.
authors.forename: Armanda Formigao
authors.initials: AF
authors.lastname: Gameiro
authors.affiliation: the Institute for Medical Microbiology, Justus-Liebig University, 35390 Giessen, Germany, and.
authors.forename: Maria
authors.initials: M
authors.lastname: Loose
authors.affiliation: the Institute for Medical Microbiology, Justus-Liebig University, 35390 Giessen, Germany, and.
authors.forename: Trinad
authors.initials: T
authors.lastname: Chakraborty
authors.affiliation: the Department of Pharmacology and Toxicology, University Vienna, 1010 Vienna, Austria.
authors.forename: Waheed
authors.initials: W
authors.lastname: Shabbir
authors.affiliation: the Department of Pharmacology and Toxicology, University Vienna, 1010 Vienna, Austria.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Aufy
authors.affiliation: the Department of Pharmacology and Toxicology, University Vienna, 1010 Vienna, Austria.
authors.forename: Rosa
authors.initials: R
authors.lastname: Lemmens-Gruber
authors.affiliation: the Department of Physiology, Emory University, Atlanta, Georgia 30322, deaton@emory.edu.
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Eaton
authors.affiliation: From the Vascular Biology Center, iczikora@augusta.edu.
authors.forename: Istvan
authors.initials: I
authors.lastname: Czikora
mesh_terms: D045744:Cell Line, Tumor; D062708:Epithelial Sodium Channel Agonists / Q000494:pharmacology*; D053503:Epithelial Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D062105:Molecular Docking Simulation; D010456:Peptides, Cyclic / Q000494:pharmacology*; D017354:Point Mutation; D000072417:Protein Domains / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D062708:Epithelial Sodium Channel Agonists; D053503:Epithelial Sodium Channels; D010456:Peptides, Cyclic; D021122:Protein Subunits; C583752:AP301 peptide
keywords: Streptococcus; epithelial sodium channel (ENaC); peptides; tumor necrosis factor (TNF); ubiquitylation (ubiquitination)