title: Prostasin interacts with the epithelial Na+ channel and facilitates cleavage of the γ-subunit by a second protease.
abstract: During maturation, the α- and γ-subunits of the epithelial Na+ channel (ENaC) undergo proteolytic processing by furin. Cleavage of the γ-subunit by furin at the consensus site γRKRR143 and subsequent cleavage by a second protease at a distal site strongly activate the channel. For example, coexpression of prostasin with ENaC increases both channel function and cleavage at the γRKRK186 site. We generated a polyclonal antibody that recognizes the region 144-186 in the γ-subunit (anti-γ43) to determine whether prostasin promotes the release of the intervening tract between the putative furin and γRKRK186 cleavage sites. Anti-γ43 precipitated both full-length (93 kDa) and furin-processed (83 kDa) γ-subunits from extracts obtained from oocytes expressing αβHA-γ-V5 channels, but only the full-length (93 kDa) γ-subunit from oocytes expressing αβHA-γ-V5 channels and either wild-type or a catalytically inactive prostasin. Although both wild-type and catalytically inactive prostasin activated ENaCs in an aprotinin-sensitive manner, only wild-type prostasin bound to aprotinin beads, suggesting that catalytically inactive prostasin facilitates the cleavage of the γ-subunit by an endogenous protease in Xenopus oocytes. As dietary salt restriction increases cleavage of the renal γ-subunit, we assessed release of the 43-mer inhibitory tract on rats fed a low-Na+ diet. We found that a low-Na+ diet increased γ-subunit cleavage detected with the anti-γ antibody and dramatically reduced the fraction precipitated with the anti-γ43 antibody. Our results suggest that the inhibitory tract dissociates from the γ-subunit in kidneys from rats on a low-Na+ diet.
authors.affiliation: Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania; and mdc4@pitt.edu.
authors.forename: Marcelo D
authors.initials: MD
authors.lastname: Carattino
authors.affiliation: Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania;
authors.forename: Gunhild M
authors.initials: GM
authors.lastname: Mueller
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York.
authors.forename: Lawrence G
authors.initials: LG
authors.lastname: Palmer
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Frindt
authors.affiliation: Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania;
authors.forename: Anna C
authors.initials: AC
authors.lastname: Rued
authors.affiliation: Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania; and.
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Hughey
authors.affiliation: Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania; and.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
mesh_terms: D000818:Animals; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D005260:Female; D045683:Furin / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D009865:Oocytes / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D017673:Sodium Chloride, Dietary / Q000008:administration & dosage; D014982:Xenopus laevis; D000099271:Prostasin
chemical_list: D053503:Epithelial Sodium Channels; D045683:Furin; D021122:Protein Subunits; D012697:Serine Endopeptidases; D017673:Sodium Chloride, Dietary; D000099271:Prostasin
keywords: amiloride; channel-activating protease 1; epithelial sodium channel; prostasin; proteases