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

Self Cite

In many epithelial tissues in the body, the rate of Na ؉ reabsorption is governed by the activity of the epithelial sodium channel (ENaC). The assembly, trafficking, and turnover of the three ENaC subunits (␣, ␤, and ␥) is complex and not well understood. Recent experiments suggest that ENaC must be proteolytically cleaved for maximal activity and may explain the discrepancies reported in prior biochemical approaches focused on quantitating the trafficking and half-life of full-length subunits. As an alternative approach to examining the dynamics of ENaC subunits, we have generated doxycycline-repressible replication-defective recombinant adenoviruses encoding individual epitope-tagged mouse ENaC subunits and expressed these in polarized MDCK I cells. Co-infection with these viruses encoding all three subunits generates robust amiloride-sensitive currents in polarized MDCK cells. Significant current was also observed in cells expressing ␣-and ␥-mENaC in the absence of ␤-mENaC. These currents did not appear to result from association with endogenous canine ␤-ENaC. Treatment of ␣␤␥-expressing cells with cycloheximide (CHX) resulted in the rapid inhibition (within 3 h) of ϳ50 -80% of the initial current; however, a sizable fraction of the initial current remained even after 6 h of CHX. By contrast, CHX addition to cells expressing only ␣-and ␥-mENaC resulted in rapid decay in current with no residual fraction. Our data suggest that ENaC channels of differing stoichiometries are differentially trafficked and degraded and provide support for the possibility that noncoordinate trafficking of ENaC subunits may function in vivo as a mechanism to modulate ENaC activity.The epithelial sodium channel (ENaC) 1 is an apical heterotetramer containing ␣, ␤, and ␥ subunits that serves as the rate-limiting step in sodium reabsorption in a number of tissues (reviewed in Ref. 1). Given the clinical importance of ENaC activity, the assembly, trafficking, and response to hormonal stimulation of ENaC subunits have been subjects of considerable study and debate. While biochemical studies from several laboratories universally report a short half-life for the total pool of newly synthesized ENaC subunits (typically 1-2 h) (2-7), there is considerable dispute regarding the stability of ENaC subunits that have reached the plasma membrane. Some studies report rapid decay of all three cell surface subunits in polarized MDCK and A6 cells (4, 5), whereas we and others have observed long half-lives of ␣-and ␥-ENaC that were biotinylated at the apical plasma membrane of A6 cells (6,8). We use the term noncoordinate regulation to describe this differential turnover of ENaC subunits and have proposed possible molecular models for this observation based on experimental results from many laboratories (6, 9).Considerable data suggest that activation of ENaC channels is regulated by proteolytic enzymes (10 -12). Recently, new evidence has emerged that suggests that activation of ENaC involves direct furin-mediated cleavage of ␣-and ␥-ENaC subunits wi...

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confidence: 66%

Section: Cell Lines and Adenoviruses-mdck Type I Cells Were Maintainementioning

confidence: 99%

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

Mohan

Bruns

Weixel³

et al. 2004

Self Cite

“…Cleavage by Subtilisin-To further examine ␣ ENaC processing, we mutated the terminal arginine (P1) in both identified serine protease (furin) cleavage sites (Arg-178 and 204) to alanines (2). This ␣ENaC construct is referred to as ␣FM.…”

Section: Mutation Of ␣ Enac Cleavage Sites Andmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Bengrine

Lis

et al. 2009

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...

“…The ENaC is also a target for a variety of post-translational modifications, including proteolytic cleavage in its ECD, which increases its activity (40). Similar to this activating mechanism, it is possible that intracellular proteases may cleave off the N-terminal part of BASIC upon maturation or application of a certain stimulus and thus lead to a more active channel compared with resting conditions.…”

Section: What Is the Mechanistic Basis For Closed State Stabilization?-mentioning

confidence: 99%

A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC)

Schmidt

Löhrer

Alsop

et al. 2016

Edited by George CarmanThe bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na ؉ channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic ␣-helical structure within its N-terminal domain. This ␣-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction.