Epithelial Na ؉ channels (ENaCs) are activated by extracellular trypsin or by co-expression with channelactivating proteases, although there is no direct evidence that these proteases activate ENaC by cleaving the channel. We previously demonstrated that the ␣ and ␥ subunits of ENaC are cleaved during maturation near consensus sites for furin cleavage. Using site-specific mutagenesis of channel subunits, ENaC expression in furin-deficient cells, and furin-specific inhibitors, we now report that ENaC cleavage correlates with channel activity. Channel activity in furin-deficient cells was rescued by expression of furin. Our data provide the first example of a vertebrate ion channel that is a substrate for furin and whose activity is dependent on its proteolysis.Epithelial Na ϩ channels are expressed in apical membranes of high resistance Na ϩ -transporting epithelia. These channels have a key role in the regulation of extracellular fluid volume, blood pressure, and airway fluid volume. ENaCs 1 are composed of three structurally related subunits, termed ␣, , and ␥, with a presumed ␣ 2  1 ␥ 1 subunit stoichiometry (1, 2), although an alternative stoichiometry has been proposed (3). Each subunit has two membrane-spanning domains that are connected by a large extracellular loop and intracellular NH 2 and COOH termini. Residues preceding and within the second membranespanning domain form the channel pore (4 -6).Previous studies have demonstrated that ENaC activity is regulated by proteases. Extracellular trypsin has been shown to increase channel activity, while extracellular serine protease inhibitors, such as aprotinin and bikunin, have been shown to decrease channel activity (7)(8)(9)(10)(11)(12). Channel activation by proteases likely reflects changes in channel gating (8,12). ENaCs characteristically have long open and closed times, generally on the order of seconds (13,14). However, a population of channels has been described that exhibit only brief (50 ms) openings and have long closed states (13,14). Caldwell et al. (12) recently reported that extracellular trypsin converts these near-silent Na ϩ channels to channels that exhibit the typically long open and closed times. A family of channel-activating serine proteases, referred to as CAPs, have been identified based on their ability to activate ENaC when co-expressed in heterologous systems (7,15,16). These serine proteases include CAP1 (or prostasin), CAP2, CAP3, and a member of a family of transmembrane serine proteases (TMPRSS3) (7,15,16). However, it is not known whether proteolysis of ENaC subunits or cleavage of a distinct regulatory protein is responsible for the activation of Na ϩ channels.We recently reported that maturation of mouse ENaC in both Chinese hamster ovary (CHO) and Madin-Darby canine kidney (MDCK) cells involves proteolytic cleavage of the ␣ and ␥ subunits (17). Expression of individual subunits revealed full-length forms of the ␣, , and ␥ subunits (95, 96, and 93 kDa, respectively) that had immature N-glycans. However, co-expression o...
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 ␣ subunit and at a single site within the ␥ subunit. We now report that the serine protease prostasin further activates ENaC by inducing cleavage of the ␥ subunit at a site distal to the furin cleavage site. Dual cleavage of the ␥ subunit is predicted to release a 43-amino acid peptide. Channels with a ␥ 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 ␥ subunit is a reversible inhibitor of endogenous ENaCs in mouse corticalcollecting duct cells and in primary cultures of human airway epithelial cells. Our results suggest that multiple proteases cleave ENaC ␥ subunits to fully activate the channel.Epithelial sodium channels (ENaC) 3 are expressed at the apical plasma membrane of cells lining the distal nephron, airway and alveoli, and distal colon, where they play a key role in the regulation of extracellular fluid volume, blood pressure, and airway surface liquid volume. These channels are composed of three homologous subunits, termed ␣, , and ␥. Each subunit has cytosolic amino and carboxyl termini and two membranespanning domains separated by a large ectodomain (1-3). The second membrane-spanning domain and the preceding region of each subunit are predicted to form the channel pore (4 -7). Proteolysis of ENaC subunit extracellular domains at specific sites has a key role in modulating channel gating (8 -10). Maturation of ENaC subunits in Xenopus oocytes, Madin-Darby canine kidney (MDCK) cells, and Chinese hamster ovary cells involves furin-dependent cleavage at two sites in the extracellular loop of the ␣ subunit and at a single site within the extracellular loop of the ␥ subunit (8). Channels that lack proteolytic processing exhibit markedly reduced activity and enhanced inhibition by external Na ϩ , a process referred to as Na ϩ selfinhibition (9). ENaC subunit cleavage by furin or exogenous trypsin relieves channels from inhibition by external Na ϩ (9, 11). We previously proposed that furin-dependent proteolysis of the ␣ subunit activates ENaC by disassociating an inhibitory domain (␣Asp-206 -Arg-231) from its effector site within the channel complex (10).Endogenous proteases other than furin likely have a role in the processing and activation of ENaC. A number of serine proteases, referred to as "channel activating proteases," have been identified that increase ENaC activity when co-expressed with ENaC in heterologous expression systems (12-14). Furthermore, selective serine protease inhibitors that do not block furin, such as aprotinin and bikunin, reduce ENaC activity (14 -20). Prostasin is an aprotinin-sensitive "channel activating (serine) protease" that inc...
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.Many ion channels are silent at rest and are activated in response to a variety of factors, including membrane potential, external ligands, and intracellular signaling processes. The ENaC 2 has evolved as a channel that is thought to reside primarily in an active state, facilitating the bulk movement of Na ϩ out of renal tubular or airway lumens. The regulated insertion and retrieval of channels at the plasma membrane have important roles in modulating ENaC-dependent Na ϩ transport (1). A number of factors also have a role in regulating ENaC activity via changes in channel P o or gating. In this regard, it has become increasingly apparent that proteolysis of ENaC subunits has a key role in this process (2). This minireview addresses several questions regarding the role of ENaC subunit proteolysis in regulating channel gating. (i) Where are ENaC subunits cleaved? (ii) Which proteases mediate ENaC cleavage? (iii) Why are channels activated by proteolysis? (iv) Is proteolysis responsible, in part, for the highly variable channel P o that has been noted for ENaC? (v) Why have ENaCs evolved as channels that require proteolysis for activation? Where Are ENaC Subunits Cleaved?Reports in the early 1980s that serine protease inhibitors reduced transepithelial Na ϩ transport across toad urinary bladder suggested that proteases have a role in activating ENaC (3). A series of studies over the past decade have confirmed that proteases activate ENaC and have begun to elucidate the mechanism by which this occurs. Following the observation that ENaC activity was significantly reduced in epithelial cells treated with aprotinin and that low concentrations of external trypsin rapidly activated ENaC in aprotinin-pretreated cells, a series of CAPs were identified that activated ENaC when coexpressed in heterologous expression systems (4 -6). Furthermore, channels with a very low P o responded to external trypsin with a dramatic increase in P o (7).What is the target of these proteases? ENaC is composed of three structurally related subunits (␣, , and ␥) that have two membrane-spanning domains connected by a large extracellular loop composed of ϳ450 residues. Early reports suggested that ENaC subunits or closely associated proteins were the protease target (5). Subsequent studies demonstrated that the ␣ and ␥ subunits of ENaC were processed by proteases (8 -11). The presence of full-length forms as well as faster migrating forms of the ␣ and ␥ subunits on SDS-polyacrylamide gels, both in cell lysates and...
The epithelial Na ؉ channel (ENaC) is a tetramer of two ␣-, one -, and one ␥-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 ␣-(95 kDa), -(96 kDa), and ␥-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of ␣␥ENaC subunits revealed a second band for each subunit (65 kDa for ␣, 110 kDa for , and 75 kDa for ␥) 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 [ 3 H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed ␣-and ␥-subunits is also consistent with proteolytic cleavage. By using ␣-and ␥-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the ␣-and ␥-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for ␣ and 18 kDa for ␥) 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.The amiloride-sensitive epithelial Na ϩ channel (ENaC) 1 is composed of three structurally related subunits, termed ␣-, -, and ␥-ENaC. The three subunits exhibit limited amino acid sequence identity (30 -40%) but are structurally similar with two membrane-spanning domains and cytosolic amino and carboxyl termini. We and others have shown that ENaC expressed in Xenopus oocytes has a subunit stoichiometry of two ␣-, one -, and one ␥-subunit (1, 2
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...
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 ␣, , and ␥, which have intracellular amino and carboxyl termini and two membranespanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the ␣ subunit and at a single site within the ␥ subunit. Epithelial sodium channels (ENaCs) 2 mediate Na ϩ entry across the apical membrane of high resistance epithelia, including the distal nephron, airway and alveolar epithelia, and distal colon. These channels are composed of three homologous subunits, termed ␣, , and ␥, which have intracellular amino and carboxyl termini and two membranespanning domains connected by large extracellular loops (1). Residues preceding and within the second membrane-spanning domain constitute the channel pore (2-5). ENaCs have a key role in the regulation of urinary Na ϩ reabsorption, extracellular fluid volume homeostasis, and control of blood pressure (6 -9). Epithelial Na ϩ channel gain-of-function mutations have been identified in patients with Liddle syndrome, a disorder characterized by volume expansion and hypertension (10 -15).In airway epithelia, ENaC has an important role in regulating the volume of airway surface liquids and mucociliary clearance. Increased ENaC activity is thought to contribute to poor mucociliary clearance observed in cystic fibrosis (16). Maturation of ENaC subunits in Xenopus oocytes, Chinese hamster ovarycells,andMadin-Darbycaninekidney(MDCK)cellsinvolvesfurindependent proteolysis at two sites within the ␣ subunit (after Arg-205 and Arg-231) and at a single site within the ␥ subunit (after Arg-143) (17, 18). Interestingly, both processed (i.e. cleaved) and unprocessed ENaCs are expressed on the cell surface (19). Proteolytic processing of ENaC likely occurs within the biosynthetic pathway as well as at the cell surface. Furin, a serine protease that resides primarily in the trans-Golgi network, is required both for cleavage and activation of ENaC in oocytes, Chinese hamster ovary, and MDCK cells (18). Other proteases, such as prostasin (also known as CAP-1) (20 -23), CAP-2 and CAP-3 (24), trypsin (18,20,25), and elastase (26), are also thought to have a role in the proteolytic processing and activation of ENaC. Proteolysis activates the channel by increasing channel open probability (25,27,28).We recently reported that ␣ subunits lacking either one or both cleavage sites exhibited a marked enhancement of the Na ϩ self-inhibition response, suggesting that ␣ subunits must be cleaved at both furin consensus sites in order to activate the channel (28). We now report that ENaCs with mutant ␣ subunits lacking either one or both furin cleavage sites exhibited markedly reduced activity, confirming that cleavage at a single furin site in the ␣ subunit was not sufficient to a...
Epithelial Na+ channels (ENaC) are inhibited by extracellular Na+, a process referred to as Na+ self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in alpha, or one site in gamma, blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary cells. We now report that channels with mutant alpha-subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the Na+ self-inhibition response, while channels with a mutant gamma-subunit showed a modestly enhanced Na+ self-inhibition response. Analysis of Na+ self-inhibition at varying [Na+] indicates that channels containing mutant alpha-subunits exhibit an increased Na+ affinity. At the single-channel level, channels with a mutant alpha-subunit had a low open probability (P(o)) in the presence of a high external [Na+] in the patch pipette. P(o) dramatically increased when trypsin was also present, or when a low external [Na+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving Na+ self-inhibition and that activation requires that the alpha-subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC P(o) and extracellular [Na+], supporting the notion that Na+ self-inhibition reflects a P(o) reduction due to high extracellular [Na+].
MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.