title: Glycosylation-dependent activation of epithelial sodium channel by solnatide.
abstract: Dysfunction of the epithelial sodium channel (ENaC), which regulates salt and water homeostasis in epithelia, causes several human pathological conditions, including pulmonary oedema. This is a potentially lethal complication of acute lung injury at least partially caused by dysfunctional alveolar liquid clearance, which in turn impairs alveolar gas exchange. Solnatide (named TIP-peptide, AP301), a 17 residue peptide mimicking the lectin-like domain of TNF has been shown to activate ENaC in several experimental animal models of acute lung injury and is being evaluated as a potential therapy for pulmonary oedema. The peptide has recently completed phase 1 and 2a clinical trials. In this study, we identify a glycosylation-dependent mechanism that preserves ENaC function and expression. Since our previous data suggested that the pore-forming subunits of ENaC are essential for maximal current activation by solnatide, we performed single- and multi-N-glycosylation site mutations in αN232,293,312,397,511Q- and δN166,211,384Q-subunits, in order to identify crucial residues for interaction with solnatide within the extracellular loop of the channel. Additionally, we generated αL576X and αN232,293,312,397,511Q,L576X deletion mutants of ENaC-α, since we have previously demonstrated that the carboxy terminal domain of this subunit is also involved in its interaction with solnatide. In cells expressing αN232,293,312,397,511Q,L576Xβγ-hENaC or δN166,311,384Q,D552Xβγ-hENaC activation by solnatide, as measured in whole cell patch clamp mode, was completely abolished, whereas it was attenuated in αL576Xβγ-hENaC- and δD552Xβγ-hENaC-expressing cells. Taken together, our findings delineate an N-glycan dependent interaction between the TIP-peptide and ENaC leading to normalization of both sodium and fluid absorption in oedematous alveoli to non-oedematous levels.
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, A-1090 Vienna, Austria; APEPTICO GmbH, A-1150 Vienna, Austria. Electronic address: waheed.shabbir@univie.ac.at.
authors.forename: Waheed
authors.initials: W
authors.lastname: Shabbir
authors.affiliation: APEPTICO GmbH, A-1150 Vienna, Austria.
authors.forename: Susan
authors.initials: S
authors.lastname: Tzotzos
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, A-1090 Vienna, Austria.
authors.forename: Minela
authors.initials: M
authors.lastname: Bedak
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, A-1090 Vienna, Austria.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Aufy
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, A-1090 Vienna, Austria.
authors.forename: Anita
authors.initials: A
authors.lastname: Willam
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, A-1090 Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Kraihammer
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, A-1090 Vienna, Austria.
authors.forename: Alexander
authors.initials: A
authors.lastname: Holzner
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Georgia Regents University, Augusta, 30912 GA, USA.
authors.forename: Istvan
authors.initials: I
authors.lastname: Czikora
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, A-1090 Vienna, Austria.
authors.forename: Parastoo
authors.initials: P
authors.lastname: Scherbaum-Hazemi
authors.affiliation: APEPTICO GmbH, A-1150 Vienna, Austria.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Fischer
authors.affiliation: APEPTICO GmbH, A-1150 Vienna, Austria.
authors.forename: Helmut
authors.initials: H
authors.lastname: Pietschmann
authors.affiliation: APEPTICO GmbH, A-1150 Vienna, Austria.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Fischer
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Georgia Regents University, Augusta, 30912 GA, USA.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Lucas
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, A-1090 Vienna, Austria.
authors.forename: Rosa
authors.initials: R
authors.lastname: Lemmens-Gruber
mesh_terms: D053503:Epithelial Sodium Channels / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D053503:Epithelial Sodium Channels; D010456:Peptides, Cyclic; C583752:AP301 peptide
keywords: Epithelial sodium channel (ENaC); Glycosylation; Patch-clamp; Western blot