Cleavage in the γ‐subunit of the epithelial sodium channel (ENaC) plays an important role in the proteolytic activation of near‐silent channels

Abstract

The mechanisms by which proteases activate the epithelial sodium channel (ENaC) are not yet fully understood. We investigated the effect of extracellular proteases on rat ENaC heterologously expressed in Xenopus laevis oocytes. Application of trypsin increased ENaC whole-oocyte currents by about 8-fold without a concomitant increase in channel surface expression. The stimulatory effect of trypsin was preserved in oocytes expressing αγ-ENaC, but was abolished in oocytes expressing αβ-ENaC. Thus, the γ-subunit appears to be essential for channel activation by extracellular proteases. Site-directed mutagenesis of a putative prostasin cleavage site in the extracellular loop of the γ-subunit revealed that mutating the 181Lys residue to alanine (γK181A) increases ENaC baseline whole-oocyte currents, decreases channel surface expression, and largely reduces the stimulatory effect of extracellular proteases (trypsin, chymotrypsin and human neutrophil elastase). In single-channel recordings from outside-out patches we demonstrated that the γK181A mutation essentially abolishes the activation of near-silent channels by trypsin, while a stimulatory effect of trypsin on channel gating is preserved. This apparent dual effect of trypsin on channel gating and on the recruitment of near-silent channels was confirmed by experiments using the β518C mutant ENaC which can be converted to a channel with an open probability of nearly one by exposure to a sulfhydryl reagent. Interestingly, the γK181A mutation results in the spontaneous appearance of a 67 kDa fragment of the γ-subunit in the plasma membrane which can be prevented by a furin inhibitor and also occurs after channel activation by extracellular trypsin. This suggests that the mutation promotes channel cleavage and activation by endogenous proteases. This would lower the pool of near-silent channels and explain the constitutive activation and reduced responsiveness of the mutant channel to extracellular proteases. We conclude that the mutated site (K181A) affects a region in the γ-subunit of ENaC that is functionally important for the activation of near-silent channels by extracellular proteases.

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

“…Our data indicate that the effects of exogenous proteases in activating ENaC expressed in oocytes are likely mediated by processing of unprocessed ␣ at the membrane and propose a dominant role of this subunit in channel activation. This conclusion is at odds with two recent reports indicating that ␥ processing is dominant in this system (5,15). However, it is important to point out that our results are not directly comparable with those of Carattino et al (15) as these authors did not examine plasma membrane ENaC subunit processing, and to Diakov et al (5), who base their conclusion of the role of ␥ by examining processing and the effects of trypsin on dimeric ␣/␤ and ␣/␥ channels.…”

“…This represents an enormous capacity for changes in ENaC activity at the plasma membrane and demonstrates the critical role of ␣ subunit cleavage in the control of activity. This dominant role of ␣ does not rule out the currently proposed role for ␥ subunit cleavage (5,15). These studies cannot be directly compared with those of ours as Diakov et al (5) examined expression of channels with two subunit combinations, whereas Carattino et al (15) did not show any comparable biochemical data.…”

“…This dominant role of ␣ does not rule out the currently proposed role for ␥ subunit cleavage (5,15). These studies cannot be directly compared with those of ours as Diakov et al (5) examined expression of channels with two subunit combinations, whereas Carattino et al (15) did not show any comparable biochemical data. Moreover, in our studies ␥ was maximally endogenously cleaved, and therefore, it could not be rate-limiting to channel activation.…”

“…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.…”

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

“…on nociceptors, which in turn activates TRPV4, allowing the influx of extracellular Ca 2ϩ ions. The residual responses in mice lacking PAR 2 may be attributable to activation of other PARs, including PAR 1 , which is also expressed by nociceptors (51) and which responds to elastase (34), or due to elastase activation of ion channels, such as the epithelial sodium channel (52,53). Residual responses in TRPV4-deficient mice may be due to PAR 2 -dependent activation of other TRPs, such as TRPV1 or TRAPA1 (7,11).…”