Prostasin-dependent activation of epithelial Na<sup>+</sup>channels by low plasmin concentrations

Svenningsen, Per; Uhrenholt, Torben Rene; Palarasah, Yaseelan; Skjødt, Karsten; Jensen, Boye L.; Skøtt, Ole

doi:10.1152/ajpregu.00321.2009

Cited by 64 publications

(79 citation statements)

References 44 publications

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“…Thus, in both cases the loss of the segment between the two furin cleavage sites is not likely to be an obligatory mechanism of ENaC activation by ␣ cleavage. Such built-in redundancy into channel cleavage would certainly be consistent with the ability of multiple diverse proteases such as trypsin, subtilisin, elastase, prostasin, furin, and plasmin to activate ENaC in different systems with a similar final outcome (9,24,(31)(32)(33)(34)(35).…”

Section: Discussionmentioning

confidence: 68%

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Bengrine

Lis

et al. 2009

Journal of Biological Chemistry

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We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of ␣ENaC using the serine protease subtilisin. Trimeric channels formed with ␣FM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an ϳ10-fold decrease in amiloride-sensitive whole cell conductance as compared with ␣WT (2.2 versus 21.2 microsiemens ( S)). Both ␣WT and ␣FM were activated to similar levels by subtilisin cleavage. Channels formed with ␣FD, a construct that deleted the segment between the two furin sites (⌬175-204), exhibited an intermediate conductance of 13.2 S. More importantly, ␣FD retained the ability to be activated by subtilisin to 108.8 ؎ 20.9 S, a level not significantly different from that of subtilisin activated ␣WT (125.6 ؎ 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of ␣ was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with ␣LD, a construct that extended the deleted sequence of ␣FD by 17 amino acids (⌬175-221). Channels with ␣LD were uncleaved, exhibited low baseline activity (4.1 S), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.It is well established that serine proteases activate the epithelial sodium channel (ENaC). 2 Activation occurs by direct mechanisms that induce channel subunit cleavage (1, 2) as well as those that are cleavage-independent but may involve cleavage of protease-activated membrane receptors (3). Channel cleavage studies have established that cellular proteases such as furin endogenously cleave the channel ␣ and ␥ subunits. Mutation of identified endogenous cleavage sites on both of these subunits diminished baseline activity, demonstrating a role for cleavage in ENaC activation.The acute effects of ENaC cleavage have largely relied on examining the effects of the protease trypsin on the ␣ and ␥ subunits. These studies have examined the effects of cleavage on wild type and furin cleavage-deficient ENaC in oocytes and epithelial cells (1,4,5). Although these have markedly improved our understanding of channel activation by serine proteases, they suffer from the main shortcoming that trypsin is a non-selective serine protease that can cleave after a single arginine residue (6, 7), and therefore, it only offers a limited tool for examining the mechanisms of cleavage at specific sites. Consistent with the reduced specificity for trypsin is the observation that ENaC retains its cleavage by this protease after mutation of consensus cleavage sites for furin (1).Despite their limitations, these studies have ind...

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Section: Discussionmentioning

confidence: 68%

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Bengrine

Lis

et al. 2009

Journal of Biological Chemistry

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“…34,35 Our previous data have shown the existence of a cell-attached cascade whereby low concentrations of plasmin (≤1 µg/mL) activates cell-anchored prostasin. 7 At the measured concentrations of active plasmin in urine, involvement of amplification, that is, prostasin, in the activation of ENaC, is likely. 7,36 Adjacent cleavage sites for plasmin and prostasin have been identified in the N terminal of γENaC.…”

Section: Discussionmentioning

confidence: 95%

“…7 At the measured concentrations of active plasmin in urine, involvement of amplification, that is, prostasin, in the activation of ENaC, is likely. 7,36 Adjacent cleavage sites for plasmin and prostasin have been identified in the N terminal of γENaC. 6 According to the stepwise proteolytic activation concept proposed by Kleyman et al, 6 plasmin and prostasin elicit extracellular second hit proteolysis subsequent to intracellular furin to release an inhibitory peptide from γENaC.…”

Section: Discussionmentioning

confidence: 95%

“…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.…”

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confidence: 99%

“…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.…”

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confidence: 99%

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Urinary Plasmin Activates Collecting Duct ENaC Current in Preeclampsia

et al. 2012

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In nephrotic syndrome, plasminogen is aberrantly filtered from plasma to the urinary space and activated along the tubular system. In vitro, plasmin increases ENaC current by proteolytic cleavage of the γ-subunit. It was hypothesized that preeclampsia is associated with plasmin-dependent ability of tubular fluid to activate ENaC. Urine was sampled from 16 preeclamptic (PE) patients and 17 normotensive pregnant women (Ctrl). Urine was analyzed for plasmin(ogen), creatinine, albumin, aldosterone, Na + , K + , proteolytic activity, and for its effect on inward current in cortical collecting duct cells (M1 cells) by whole-cell patch clamp. In PE, urine plasmin(ogen): creatinine ratio was elevated 40-fold (geometric mean, 160 versus 4 µg/g; P <0.0001) and urine aldosterone: creatinine ratio was suppressed to 25% of Ctrl (geometric mean, 27 versus 109 µg/g; P <0.001). A significant negative correlation was found in PE between urinary plasmin(ogen) and aldosterone ( P <0.05). In PE, proteolytic activity was detected at 90 to 75 kD by gelatin zymography in 14 of 16 patients and confirmed by serine protease assay. Immunoblotting showed active plasmin in PE urine. Whole-cell inward current increased in M1 cells on exposure to urine from PE (173±21%; n=6; P <0.001). The increase in current was abolished by amiloride (2 μmol/L; P <0.001), α 2 -antiplasmin (1 μmol/L; P <0.001), and heat denaturation ( P <0.001). Preeclampsia is associated with urinary excretion of plasmin(ogen) and plasmin-dependent activation of ENaC by urine. Proteolytic activation of ENaC by plasmin may contribute to Na + retention and hypertension in preeclampsia.

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