Prostasin interacts with the epithelial Na+ channel and facilitates cleavage of the [gamma]-subunit by a second protease

• Published in: American journal of physiology. Renal physiology 2014-11, Vol.307 (9), p.F1080
• Main Authors: Carattino, Marcelo D, Mueller, Gunhild M, Palmer, Lawrence G, Frindt, Gustavo, Rued, Anna C, Hughey, Rebecca P, Kleyman, Thomas R
• Format: Article
• Language: English
• Subjects: Cells; Gene expression; Kidneys; Maturation; Polyclonal antibodies; Proteases; Proteins
• ISSN: 1931-857X; 1522-1466

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 γRKRR... 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 γRKRK... 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 γRKRK... 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. (ProQuest: ... denotes formulae/symbols omitted.)