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.

Sodium Retention and Volume Expansion in Nephrotic Syndrome: Implications for Hypertension

“…Evidence for this comes from recent studies that employed an antibody raised against the γ subunit inhibitory tract. Sodium restriction lead to loss of the inhibitory tract from the C-terminal fragment, consistent with proteolytic removal of the inhibitory tract and channel activation 44 .…”

“…Interestingly, prostasin does not require its proteolytic activity to activate ENaC 44,64 . Prostasin is present within a protein complex that contains ENaC, and recent work suggests that prostasin can recruit other proteases to this complex, resulting in channel cleavage and activation 44 . Svenningsen et al have suggested that prostasin facilitates the targeting of plasmin to ENaC at the plasma membrane 63 .…”

Association of Plasminuria with Overhydration in Patients with CKD

“…It is an emerging concept that serine proteases contribute to ENaC regulation by cleaving specific sites in the extracellular domains of the a-and g-subunits (6)(7)(8)(9)(10), resulting in the release of inhibitory peptides and activation of the channel (11)(12)(13). Proteolytic activation of ENaC by serine proteases as shown in native renal tissue (14) is enhanced by low-salt diet or aldosterone infusion (15) and might involve membrane-anchored prostasin (16) and/or tissue (urinary) kallikreins (17). Under pathophysiologic conditions of proteinuria, ENaC is thought to be aberrantly activated by plasmin that arises from filtered plasminogen and conversion by the tubular urokinase-type plasminogen activator (4,6).…”

Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels

“…Then, anti-V5 or anti-HA monoclonal ) antibodies were added to the samples (1:5,000 and 1:1,000 dilution, respectively). Our and other groups have demonstrated that non-ENaC bands could be detected in parental oocytes or oocytes expressing ENaC constructs without these artificial flags (Carattino et al, 2014;Ji, Zhao, Komissarov, Chang, Liu & Matthay, 2015;Passero, Mueller, Myerburg, Carattino, Hughey & Kleyman, 2012;Passero, Mueller, Rondon-Berrios, Tofovic, Hughey & Kleyman, 2008). Horseradish peroxidase-labelled secondary antibodies (Jackson Immunoresearch) were used (1:10,000).…”