title: Cell-surface expression of the channel activating protease xCAP-1 is required for activation of ENaC in the Xenopus oocyte.
abstract: Sodium balance, extracellular fluid volume, and ultimately BP are maintained by precise regulation of the activity of the epithelial sodium channel (ENaC). Using a functional complementation assay in the Xenopus laevis oocyte expression system, a channel-activating protease (CAP-1) that increases ENaC activity two to threefold in the Xenopus oocyte expression system is here identified. External application of trypsin mimics the effect of Xenopus CAP-1 (xCAP-1) on ENaC activity, which can be blocked by aprotinin, a serine protease inhibitor, suggesting the existence of a novel extracellular pathway for controlling ENaC activity. Sequence analysis predicts that CAP-1 is a secreted and/or glycosyl-phosphatidyl-inositol (GPI)-anchored protein. The aim of the present study was to determine whether cell-surface expression of xCAP-1 is required for ENaC activation. By site-directed mutagenesis of xCAP-1, the importance of the catalytic site, N-glycosylation, and the GPI anchor of xCAP-1 on ENaC activity were analyzed. Glycosylation or catalytic activity is not required for cell-surface expression of xCAP-1, whereas the deletion of the GPI anchor consensus motif at the C-terminus of xCAP-1 (G305Stop) abolishes cell-surface expression and ENaC activation. G305Stop-mutated xCAP-1 is recovered as a secreted protein in the external medium. A catalytic mutant of xCAP-1 significantly decreased ENaC activation but did not fully abolish the effect of xCAP-1. The data indicate the critical role of the GPI anchor in ENaC activation and suggest that catalytic and noncatalytic mechanisms are involved.
authors.affiliation: *Institute of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and Institute of Anatomy, University of Zurich, Zurich, Switzerland.
authors.forename: Véronique
authors.initials: V
authors.lastname: Vallet
authors.affiliation: *Institute of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and Institute of Anatomy, University of Zurich, Zurich, Switzerland.
authors.forename: Corinne
authors.initials: C
authors.lastname: Pfister
authors.affiliation: *Institute of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and Institute of Anatomy, University of Zurich, Zurich, Switzerland.
authors.forename: Johannes
authors.initials: J
authors.lastname: Loffing
authors.affiliation: *Institute of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and Institute of Anatomy, University of Zurich, Zurich, Switzerland.
authors.forename: Bernard C
authors.initials: BC
authors.lastname: Rossier
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D053503:Epithelial Sodium Channels; D005260:Female; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000502:physiology; D009865:Oocytes / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015222:Sodium Channels / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D053503:Epithelial Sodium Channels; D017261:Glycosylphosphatidylinositols; D015222:Sodium Channels; D012697:Serine Endopeptidases; C452639:xCAP-1