Rebecca P. Hughey

Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit.

Epithelial sodium channels (ENaC) are expressed in the apical membrane of high resistance Na+ transporting epithelia and have a key role in regulating extracellular fluid volume and the volume of airway surface liquids. Maturation and activation of ENaC subunits involves furin-dependent cleavage of the ectodomain at two sites in the α subunit and at a single site within the γ subunit. We now report that the serine protease prostasin further activates ENaC by inducing cleavage of the γ subunit at a site distal to the furin cleavage site. Dual cleavage of the γ subunit is predicted to release a 43-amino acid peptide. Channels with a γ subunit lacking this 43-residue tract have increased activity due to a high open probability. A synthetic peptide corresponding to the fragment cleaved from the γ subunit is a reversible inhibitor of endogenous ENaCs in mouse cortical-collecting duct cells and in primary cultures of human airway epithelial cells. Our results suggest that multiple proteases cleave ENaC γ subunits to fully activate the channel.

Maturation of the Epithelial Na+ Channel Involves Proteolytic Processing of the α- and γ-Subunits

The epithelial Na+ channel (ENaC) is a tetramer of two α-, one β-, and one γ-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone α-(95 kDa), β-(96 kDa), and γ-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of αβγENaC subunits revealed a second band for each subunit (65 kDa for α, 110 kDa for β, and 75 kDa for γ) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed α- and γ-subunits is also consistent with proteolytic cleavage. By using α- and γ-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the α- and γ-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for α and 18 kDa for γ) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.

ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases

Epithelial Na+ channels facilitate the transport of Na+ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na+ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.

Plasmin Activates Epithelial Na+ Channels by Cleaving the γ Subunit

Proteolytic processing of epithelial sodium channel (ENaC) subunits occurs as channels mature within the biosynthetic pathway. The proteolytic processing events of the α and γ subunits are associated with channel activation. Furin cleaves the α subunit ectodomain at two sites, releasing an inhibitory tract and activating the channel. However, furin cleaves the γ subunit ectodomain only once. A second distal cleavage in the γ 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 γ subunit at γLys194, a site distal to the furin site. A γK194A mutant prevented both plasmin-dependent activation of ENaC and plasmin-dependent production of a unique 70-kDa carboxyl-terminal γ 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.

Characterization and physiological function of rat renal gamma-glutamyltranspeptidase.

Rat renal gamma-glutamyltranspeptidase is an intrinsic membrane glycoprotein. The larger of its two subunits is apparently folded into two distinguishable domains which are separated by a protease-sensitive sequence of amino acids. Membrane binding of gamma-glutamyltranspeptidase results from the hydrophobic interaction of the nonpolar domain of the amphipathic subunit with the lipid bilayer. Localization of at least a portion of the gamma-glutamyl binding site on the smaller subunit limits the active site of the enzyme to one side of the membrane. Within the kidney, the enzyme is primarily associated with the luminal surface of the brush border membrane of the proximal straight tubule. Comparison of the kinetic properties of gamma-glutamyltranspeptidase with the pH and the substrates available within the tubular fluid suggests that the physiologically significant reaction catalyzed by the transpeptidase is the hydrolysis of glutathione and its S-derivatives. The glutathionemia and glutathionuria observed in a patient who lacks detectable gamma-glutamyltranspeptidase activity and in mice following specific inhibition of transpeptidase, support the hypothesis that the enzyme plays a major role in glutathione catabolism. It now appears that the activities attributed to the gamma-glutamyl cycle do not participate in amino acid transport, but instead constitute three separate metabolic pathways; the intracellular synthesis of glutathione, the intracellular degradation of gamma-glutamyl peptides and the extracellular hydrolysis of glutathione. The finding that various cells release reduced and oxidized glutathione indicates that glutathione turnover may be a process of intracellular synthesis, excretion and extracellular degradation.

Specificity of a particulate rat renal peptidase and its localization along with other enzymes of mercapturic acid synthesis

Abstract Renal processing of S -derivatized glutathiones to mercapturic acids requires the participation of three enzymatic activities: γ-glutamyl hydrolase or transpeptidase, a peptidase which is capable of hydrolyzing S -derivatized cysteinylglycine, and an N -acetyltransferase. A particulate peptidase, which was assayed with S -benzylcysteine- p -nitroanilide, was found to be localized along with γ-glutamyltranspeptidase and N -acetyltransferase in the outer stripe region of the renal medulla. This localization suggests that these three activities may be contained primarily in the proximal straight tubules. Results of differential and isopycnic centrifugation indicate that the particulate peptidase is contained along with γ-glutamyltranspeptidase in the brush border membranes while the N -acetyltransferase is probably associated with the endoplasmic reticulum. The partially purified peptidase (200-fold) exhibits a broad substrate specificity. It has greater activity with reduced than oxidized cysteinylglycine, but S -derivatized substrates are hydrolyzed even faster. Comparison of its activity with various substrates indicates that it prefers peptides with a hydrophobic N -terminal amino acid and that it may require a free amino group. Heat-inactivation studies suggest that all of these activities are attributable to a single enzyme. These results suggest that this peptidase may participate along with γ-glutamyltranspeptidase and an N -acetyltransferase in the conversion of glutathione conjugates to mercapturic acids.

The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its alpha subunit.

Epithelial sodium channels (ENaCs) mediate Na+ entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed α, β, and γ, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the α subunit and at a single site within the γ subunit. The α subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking α subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both α subunit cleavage and the tract αAsp-206-Arg-231 are active when expressed in oocytes, suggesting that αAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (α-26), corresponding to αAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na+ channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC50 for amiloride block of ENaC was not affected by the presence of α-26, indicating that α-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within α-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of α-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the α subunit activates ENaC by disassociating an inhibitory domain (αAsp-206-Arg-231) from its effector site within the channel complex.

Proteolytic processing of the epithelial sodium channel gamma subunit has a dominant role in channel activation.

Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within the α subunit and at a single extracellular site within the γ subunit. Channels lacking furin processing of the α subunit have very low activity. We recently identified a prostasin-dependent cleavage site (RKRK186) in the γ subunit. We also demonstrated that the tract α D206-R231, between the two furin cleavage sites in the α subunit, as well as the tract γ E144-K186, between the furin and prostasin cleavage sites in the γ subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of the α and γ subunit inhibitory tracts, respectively. We examined whether release of either theα orγ inhibitory tract has a dominant role in activating the channel. Co-expression of prostasin and either wild type channels or mutant channels lacking furin cleavage of the α subunit (αR205A,R208A,R231Aβγ) in Xenopus laevis oocytes led to increases in whole cell currents to similar levels. In an analogous manner and independent of the proteolytic processing of theα subunit, amiloride-sensitive currents in oocytes expressing channels carrying γ subunits with both a mutation in the furin cleavage site and a deletion of the inhibitory tract (αβγR143A,ΔE144-K186 and αR205A,R208A,R231AβγR143A, ΔE144-K186) were significantly higher than those from oocytes expressing wild type ENaC. When channels lacked the α and γ subunit inhibitory tracts, α subunit cleavage was required for channels to be fully active. Channels lacking both furin cleavage and the inhibitory tract in theγ subunit (αβγR143A,ΔE144-K186) showed a significant reduction in the efficacy of block by the syntheticα-26 inhibitory peptide representing the tract αD206-R231. Our data indicate that removal of the inhibitory tract from the γ subunit, in the absence ofα subunit cleavage, results in nearly full activation of the channel.

Role of Epithelial Sodium Channels and Their Regulators in Hypertension

The kidney has a central role in the regulation of blood pressure, in large part through its role in the regulated reabsorption of filtered Na+. Epithelial Na+ channels (ENaCs) are expressed in the most distal segments of the nephron and are a target of volume regulatory hormones. A variety of factors regulate ENaC activity, including several aldosterone-induced proteins that are present within an ENaC regulatory complex. Proteases also regulate ENaC by cleaving the channel and releasing intrinsic inhibitory tracts. Polymorphisms or mutations within channel subunits or regulatory pathways that enhance channel activity may contribute to an increase in blood pressure in individuals with essential hypertension.

Role of proteolysis in the activation of epithelial sodium channels.

Purpose of reviewEpithelial sodium channels mediate Na+ transport across high resistance, Na+-transporting epithelia. This review describes recent findings that indicate that epithelial sodium channels are activated by the proteolytic release of inhibitory peptides from the α and γ subunits. Recent findingsNon-cleaved channels have a low intrinsic open probability that may reflect enhanced channel inhibition by external Na+ – a process referred to as Na+ self-inhibition. Cleavage at a minimum of two sites within the α or γ subunits is required to activate the channel, presumably by releasing inhibitory fragments. The extent of epithelial sodium channel proteolysis is dependent on channel residency time at the plasma membrane, as well as on the balance between levels of expression of proteases that activate epithelial sodium channels and inhibitors of these proteases. Regulated epithelial sodium channel proteolysis has been observed in rat kidney and in human airway epithelia. SummaryProteolysis of epithelial sodium channel subunits plays a key role in modulating epithelial sodium channel activity through changes in channel open probability.