Indirect Activation of the Epithelial Na+ Channel by Trypsin

Bengrine, Abderrahmane; Li, Jinqing; Hamm, L. Lee; Awayda, Mouhamed S.

doi:10.1074/jbc.m611829200

Cited by 29 publications

(35 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Such a mechanism would be consistent with that observed for ENaC activation by a variety of serine proteases in oocytes as well as epithelial cells (3,8,24). To further confirm this mechanism for subtilisin we examined its effects on ENaC surface expression in intact oocytes.…”

Section: Mutation Of ␣ Enac Cleavage Sites Andmentioning

confidence: 70%

“…Oocyte Homogenization and Western Blotting-Injected oocytes were processed as previously described (3,13). Oocytes were biotinylated on ice using a sulfo-NHS-biotin linker (Pierce) and homogenized at 10 l/oocyte in buffer containing 170 mM NaCl, 10 mM Tris, 5 mM EDTA, pH 7.5, and a protease inhibitor mixture (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…Recordings were carried out 1-3 days after injection. Procedures to determine surface binding utilized a colorimetric assay with a horseradish peroxidase-coupled anti-HA antibody (Roche Applied Science) and were previously described (3,12).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

Bengrine

Lis

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Section: Mutation Of ␣ Enac Cleavage Sites Andmentioning

confidence: 70%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Bengrine

Lis

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Trypsin and elastase are serine proteases, known to activate ENaC channels in oocytes, human bronchial cell lines, and rat alveolar epithelial cells (9,46), either by activating near-silent channels that are already present in the membrane (28), or by activating signal transduction pathways (7,47). In our experimental model, the effect of A1AT on ENaC in oocytes was not immediately reversible; only 40% of the initial current was restored 6 hours after A1AT washout from the bathing medium, indicating that a fraction of the inhibition was irreversible at least within this time frame.…”

Section: Discussionmentioning

confidence: 99%

α₁-Antitrypsin Inhibits Epithelial Na⁺ Transport In Vitro and In Vivo

Lazrak¹,

Nita²,

Subramaniyam³

et al. 2009

Am J Respir Cell Mol Biol

View full text Add to dashboard Cite

A variety of studies have shown that Na 1 reabsorption across epithelial cells depends on the protease-antiprotease balance. Herein, we investigate the mechanisms by which a 1 -antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloridesensitive epithelial Na 1 channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml 5 1 mM) decreased ENaC currents across Xenopus laevis oocytes injected with human a,b,g-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC singlechannel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO 2 ), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO 2 -treated A1AT (1 mM) in C57BL/6 mice also decreased Na 1 -dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO 2 -treated A1AT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that A1AT may be an important modulator of ENaC activity and of Na 1 -dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.Keywords: alveolar fluid clearance; serine proteases; H441 cells; Xenopus oocytes; ENaCThe amiloride-sensitive epithelial Na 1 channels (ENaCs) play an important role in Na 1 and fluid homeostasis across a number of epithelial cells, including those in airway and alveolar spaces (1, 2). ENaC mRNA, protein levels, and ENaC activity are regulated by a variety of hormones (such as aldosterone and dexamethasone), second messengers cAMP/protein kinase A, and reactive intermediates (2-4). In addition, endogenous channelactivating proteases (CAPs), as well as proteases released by inflammatory cells (trypsin, elastase), activate ENaC either by cleaving critical amino acids in a-and g-ENaC subunits, or by activating signaling pathways (1, 2, 5-7).There has been considerable interest in identifying the mechanisms by which endogenous and exogenous proteases activate ENaC. Aprotinin, a potent and reversible Kunitz-type inhibitor of several serine proteases, including trypsin, plasmin, and kallikreins (8), has been reported to inhibit sodium transport among a variety of epithelial cells, including A6 (9), human bronchial epithelial (HBE) cells (10, 11), and rat and mouse lung alveolar epithelial cells (1). Other Kunitz-type serine protease inhibitors, such as hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 (plac...

show abstract

“…ENaC is expressed in a variety of tissues including the renal collecting duct, the distal colon, the lungs and the ducts of exocrine glands. Activity of ENaC in these tissues is regulated by an array of physiological factors including hormones, nucleotides, and cytosolic ion concentrations, many of which are known to exert their effect on the channel by mechanisms that involve trimeric G proteins (12)(13)(14)(15)(16). We have previously reported, in salivary duct cells, that ENaC is maintained in an active state by the kinase activity of GRK2 and that GRK2 inhibits Na ϩ feedback inhibition of ENaC (17).…”

mentioning

confidence: 99%

Regulation of the Epithelial Na+ Channel by the RH Domain of G Protein-coupled Receptor Kinase, GRK2, and Gαq/11

Lee

Song

Campbell

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with ␣-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na ؉ absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant ( K220R GRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (⌬RH-GRK2) or a GRK2 mutant that cannot interact with G␣q/11/14 ( D110A GRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the ␣-subunits of Gq/11. We further found that expression of constitutively active G␣q/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against G␣q/11 increases ENaC activity. The effect of G␣q on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 ␣-subunits regulate the activity ENaC.The ␤-adrenergic receptor kinase 1 (GRK2) is one of the G protein-coupled receptor serine/threonine kinases (GRKs). 3All seven members of the GRK family (GRK1-7) share a highly homologous kinase domain that is flanked toward the C-terminal by a pleckstrin homology (PH) domain and toward the N-terminal by a regulator of G-protein signaling homology (RH) domain (1). Protein kinases of this family are unique in their ability to specifically phosphorylate the agonist-activated form of heptahelical G protein-coupled receptors (GPCRs) (2). Upon stimulation, GRKs facilitate binding of agonist-activated GPCRs to cytosolic cofactor proteins, arrestins, and other proteins involved in receptor desensitization (3,4). This interaction impairs coupling between the GPCRs and trimeric G proteins, targets GPCRs for clathrin-mediated endocytosis (3), and terminates G protein signal transduction (2). Activity of GRKs is, therefore, important for arbitrating an appropriate strength and duration of cellular responses, allowing the G protein-dependent cellular responses to physiological stimulation to cease rapidly after receptor activation even in the continuing presence of stimuli.Recent studies suggest that, in addition to their ability to inactivate GPCRs by phosphorylation, GRKs can phosphorylate and regulate activity of an array of non-receptor substrates (2) and also regulate GPCR signaling by a mechanism that does not require their intrinsic kinase activity (3). For instance, the RH domain of GRK2 contains a binding site th...

show abstract

Indirect Activation of the Epithelial Na+ Channel by Trypsin

Cited by 29 publications

References 52 publications

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

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

α₁-Antitrypsin Inhibits Epithelial Na⁺ Transport In Vitro and In Vivo

Regulation of the Epithelial Na+ Channel by the RH Domain of G Protein-coupled Receptor Kinase, GRK2, and Gαq/11

Contact Info

Product

Resources

About

Indirect Activation of the Epithelial Na+ Channel by Trypsin

Cited by 29 publications

References 52 publications

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

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

α1-Antitrypsin Inhibits Epithelial Na+ Transport In Vitro and In Vivo

Regulation of the Epithelial Na+ Channel by the RH Domain of G Protein-coupled Receptor Kinase, GRK2, and Gαq/11

Contact Info

Product

Resources

About

α₁-Antitrypsin Inhibits Epithelial Na⁺ Transport In Vitro and In Vivo