Plasmin Activates Epithelial Na+ Channels by Cleaving the γ Subunit

Abstract

Proteolytic processing of epithelial sodium channel (ENaC) subunits occurs as channels mature within the biosynthetic pathway. The proteolytic processing events of the ␣ and ␥ subunits are associated with channel activation. Furin cleaves the ␣ subunit ectodomain at two sites, releasing an inhibitory tract and activating the channel. However, furin cleaves the ␥ subunit ectodomain only once. A second distal cleavage in the ␥ subunit induced by other proteases, such as prostasin and elastase, is required to release a second inhibitory tract and further activate the channel. We found that the serine protease plasmin activates ENaC in association with inducing cleavage of the ␥ subunit at ␥Lys 194 , a site distal to the furin site. A ␥K194A mutant prevented both plasmin-dependent activation of ENaC and plasmin-dependent production of a unique 70-kDa carboxyl-terminal ␥ subunit cleavage fragment. Plasmin-dependent cleavage and activation of ENaC may have a role in extracellular volume expansion in human disorders associated with proteinuria, as filtered plasminogen may be processed by urokinase, released from renal tubular epithelium, to generate active plasmin.The epithelial sodium channel (ENaC) 3 transports Na ϩ across the apical membrane of principal cells in the aldosterone-sensitive distal nephron (1). Alterations in ENaC activity disrupt Na ϩ balance, leading to changes in both extracellular volume and blood pressure. Expansion of extracellular volume occurs in a variety of clinical disorders, including nephrotic syndrome. The role of ENaC activation in many of the clinical disorders associated with extracellular volume expansion remains to be defined.ENaC activity in the cells that line the distal nephron depends on the number of channels in the apical membrane and on channel open probability (P o ) (1). ENaC subunits undergo post-translational processing by specific proteases (2-9). Cleavage of the ␣ and ␥ subunits by proteases has a key role in activating ENaC, presumably by releasing inhibitory domains within the ectodomains of the ␣ and ␥ subunits (3, 5, 10, 11). We have proposed that multiple proteolytic cleavage events lead to a stepwise activation of ENaC, reflected in a stepwise increase in channel P o (3,5,10,12). Channels that lack proteolytic processing have a low P o (3, 10, 12, 13). Channels that have been cleaved solely by furin, where an ␣ subunit inhibitory tract has been released, exhibit an intermediate P o (3,10,12). Furinprocessed channels likely represent the channels that are observed in Xenopus oocytes at a single channel level. Channels that have released both ␣ and ␥ subunit inhibitory tracts exhibit a high P o , as we observed in oocytes co-expressing ENaC and prostasin (3, 5). Both non-cleaved channels and furin-processed channels at the plasma membrane provide a reservoir of channels that can be activated by extracellular proteases (3,14).One potential activator of ENaC is the serine protease plasmin. Although known for its involvement in fibrinolysis, plasmin has been implicate...

Inhibitory Tract Traps the Epithelial Na+ Channel in a Low Activity Conformation

“…Cleavage likely occurs during channel maturation by proteases in the biosynthetic pathway, as well as at the cell surface (3). ENaC proteolysis has been suggested to play a role in disorders such as nephrotic syndrome and cystic fibrosis, where concentrations of specific protease are elevated in the extracellular milieu (4,5). Activation requires cleavage at multiple sites within the ␣ and/or ␥ subunits with the liberation of imbedded inhibitory tracts (3,6,7).…”

Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites

“…Putative cleavage sites in ␥ENaC have been described for the serine proteases prostasin (9), plasmin (8,10), elastase (11), kallikrein (12), and chymotrypsin (8). In contrast, the sites at which trypsins cleave and activate ENaC have not yet been identified.…”

Urinary Plasmin Activates Collecting Duct ENaC Current in Preeclampsia

“…4,5 In vitro, inward amiloride-sensitive current in collecting duct cells is enhanced by exposure to nephrotic urine containing plasmin and by purified plasmin. 4,6 Plasmin was shown to release an inhibitory peptide tract from the exodomain of the γENaC subunit 4,6 either by direct cleavage at high concentrations or through Glycosylphophatidylinositol-anchored prostasin at low concentrations. 4,6,7 Addition of α 2 -antiplasmin and aprotinin inhibited the ability of nephrotic urine and plasmin to evoke current.…”

“…4,6 Plasmin was shown to release an inhibitory peptide tract from the exodomain of the γENaC subunit 4,6 either by direct cleavage at high concentrations or through Glycosylphophatidylinositol-anchored prostasin at low concentrations. 4,6,7 Addition of α 2 -antiplasmin and aprotinin inhibited the ability of nephrotic urine and plasmin to evoke current. 4,6,7 The in vitro cleavage of γENaC is consistent with a distinct shift in migratory pattern of renal tissue γENaC to lower molecular weight isoforms on SDS-PAGE gels in proteinuric conditions.…”

“…4,6,7 Addition of α 2 -antiplasmin and aprotinin inhibited the ability of nephrotic urine and plasmin to evoke current. 4,6,7 The in vitro cleavage of γENaC is consistent with a distinct shift in migratory pattern of renal tissue γENaC to lower molecular weight isoforms on SDS-PAGE gels in proteinuric conditions. 8,9 Sodium retention and ascites formation is mitigated by the ENaC blocker amiloride in experimental nephrotic syndrome in rats 1,4 and in children with nephrotic syndrome.…”