Raising the volume on near‐silent epithelial Na+ channels

The epithelial sodium channel (ENaC) is an apical membrane ion channel that is the rate-limiting step for sodium absorption in a number of epithelia including, but not limited to the colon, kidney and lung (Rossier, 2004). Derangement of ENaC regulation is contributory to several diseases. For example, abnormal cleavage of ENaC in cystic fibrosis airways where extracellular proteases are increased results in dangerously high levels of ENaC activation, leading to mucus stasis (Tarran et al. 2006; Myerburg et al. 2008). ENaC is a member of the ENaC/degenerin family of ion channels that are comprised of three subunits termed α, β and γ. The crystal structure of a closely related member of this family, ASIC1, has recently been solved; ASIC1 is a heterotrimer and each subunit is comprised of short amino and carboxyl termini, two membrane-spanning domains and a very large extracellular loop which appears to be enzymatically cleaved (Jasti et al. 2007). In a paper published in this issue of The Journal of Physiology, Diakov et al. (2008) have found that a single residue in the extracellular loop of the γ subunit of ENaC is important for the conversion of inactive ENaCs, which are termed near-silent channels, into active channels. ENaC has multiple modes of regulation and is sensitive to lipids, cAMP and proteases (Stutts et al. 1995; Vallet et al. 1997; Ma et al. 2002). The number of ENaCs in the membrane (N), and their open probability (Po) can both be affected during its regulation. A major mode of ENaC regulation is by proteolytic cleavage both by intracellular furin-type convertases and extracellular serine-type proteases such as trypsin, which is required for ENaC to conduct Na+. Rossier and colleagues initially identified a membrane-bound serine protease (termed channel activating protease or CAP1) that had previously been called prostasin (Vallet et al. 1997). The activation of ENaC by CAP1 was mimicked by external addition of trypsin, indicating that both CAP1 and trypsin act via the same pathway. Serine proteases can activate ENaC by increasing Po by up to eightfold (Vallet et al. 1997). ENaC can reside in the plasma membrane as both active channels with Po values close to 0.5 and near-silent channels which have Po values of less than 0.05 which can be converted to active channels following application of trypsin or another neutrophil elastase (Caldwell et al. 2005). ENaC is also partially activated by endogenous intracellular convertases such as furin (Hughey et al. 2004). However, in the Xenopus laevis oocyte expression system, exogenous trypsin or co-expression of a CAP such as prostasin is required to fully activate ENaC (Vallet et al. 1997). When ENaC is proteolytically cleaved, smaller fragments of ENaC can be detected biochemically although how proteolysis actually increases ENaC activity remains contentious. When expressing ENaC subunits in Xenopus oocytes, Diakov et al. (2008) demonstrated that αβγ or αγ but not αβ subunit combinations preserved sensitivity to extracellular proteases. The authors then focused in on a putative serine protease cleavage site that was only present in the extracellular loop of the γ ENaC subunit and found that a single point mutation at position 181 from the charged amino acid lysine to the non-charged amino acid alanine (γK181A) removed the stimulatory effect of extracellular proteases and prevented the recruitment of near-silent channels into active channels. This mutation appeared to be highly specific to position 181 and a similar mutation to an alanine at position 180 in the same subunit had no such effect. A careful and thorough electrophysiological characterization by Diakov et al. (2008) revealed that the γK181A mutation, in addition to decreasing extracellular protease sensitivity, also slightly reduced ENaC surface expression by ∼30 % yet paradoxically increased basal ENaC currents. This mutation did not change the single channel conductance. However, the authors found that the γK181A mutant increased the mean open time of the existing channels which explains the increased basal currents. To further investigate this phenomenon, the authors used the βS518C ENaC mutant which when co-expressed with wild-type α and γ ENaC subunits and exposed to the sulfhydryl reagent MTSET, rapidly increases Po to ∼1. Using α, βS518C, γ ENaC and MTSET they found that trypsin still increased the number of channels in the patch, presumably by recruiting near-silent channels. In contrast, when they combined the MTSET mutant with their novel γ mutant (α,βS518C,γK181A ENaC), the Po still increased to 1 in the presence of MTSET, but trypsin failed to recruit near-silent channels, indicating that serine proteases can affect both gating and N, and that these two processes can be uncoupled. Diakov et al. (2008) went on to investigate how proteases can affect ENaC cleavage biochemically by performing Western blots of γ ENaCs tagged with a V5 epitope on the C terminus. Using this probe, full length γ ENaC has a molecular weight of ∼87 kDa, yet the predominant band in oocytes is a 76 kDa band, which is thought to represent ENaC that has been cleaved by the ubiquitous intracellular convertase furin. Extensive work on the effect of furin on ENaC has been performed and each subunit has a furin consensus site at position 138 in the extracellular loop which, when mutated from arginine to alanine, prevents furin cleavage (Hughey et al. 2004). Trypsin pre-treatment further diminishes the size of ENaC and results in a smaller 67 kDa band. Interestingly, in oocytes expressing α,β,γK181A ENaC, the 67 kDa band was present even in the absence of extracellular trypsin, suggesting that this mutant was spontaneously cleaved and it is likely that this mutation increases the specificity of γ ENaC for endogenous proteases. However, when the furin-insensitive mutation was introduced into γ ENaC along with the γK181A mutation (α,β,γP138A,K181A ENaC) the gain of function associated with the γK181A mutation alone was absent, indicating that cleavage at the furin site must occur before cleavage at position 181 by serine proteases. Thus, these data suggest that ENaC must be cleaved by both furin-like convertases and serine proteases before near-silent ENaC channels can be fully activated (Diakov et al. 2008). ENaC regulation represents a critical step in moderating Na+ absorption in many tissues, which probably explains why ENaC has so many modes of regulation to permit tissue-specific fine tuning of this event. Knowing where ENaC is cleaved, and how it affects ENaC gating, contributes greatly to understanding the complex regulation of this channel and possibly the next step will be to understand why near-silent ENaCs must be both intracellularly and extracellularly cleaved to be activated.