Epithelial Sodium Channels Are Activated by Furin-dependent Proteolysis

Abstract

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...

Differential Current Decay Profiles of Epithelial Sodium Channel Subunit Combinations in Polarized Renal Epithelial Cells

“…Anti-V5 antibody detected two major bands that migrated close to the 97-kDa molecular mass standard in cells expressing only ␣-mENaC, similar to the pattern observed previously (13). A minor, faster migrating band was also detected that is larger than the reported 65-kDa furin-cleaved fragment of the ␣ subunit (13,14). When ␥-mENaC was immunoprecipitated from AV-infected MDCK I cells, we observed a major band of ϳ93 kDa, with a minor form migrating at ϳ75 kDa, consistent with that of furincleaved ␥-mENaC (13,14).…”

“…Recently, new evidence has emerged that suggests that activation of ENaC involves direct furin-mediated cleavage of ␣-and ␥-ENaC subunits within the biosynthetic pathway (13,14). These results suggest that changes in biochemical levels of full-length ENaC subunits may not correlate directly with modulation of sodium currents.…”

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

“…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).…”

“…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.…”

Proprotein convertase furin/PCSK3 and atherosclerosis: New insights and potential therapeutic targets

“…Mutation of furin cleavage sites results in the inhibition of ENaC cleavage and its activity in Xenopus oocytes. Further analysis showed that other members of PCs family, including PACE4, PC5/PC6 and PC7, only have a moderate impact on the ENaC maturation [55]. Taken together, these results suggest that furin-induced ENaC activation may help lower BP level.…”