Prostasin interacts with the epithelial Na+ channel and facilitates cleavage of the γ-subunit by a second protease.

During maturation, the α- and γ-subunits of the epithelial Na+ channel (ENaC) undergo proteolytic processing by furin. Cleavage of the γ-subunit by furin at the consensus site γRKRR143 and subsequent cleavage by a second protease at a distal site strongly activate the channel. For example, coexpression of prostasin with ENaC increases both channel function and cleavage at the γRKRK186 site. We generated a polyclonal antibody that recognizes the region 144-186 in the γ-subunit (anti-γ43) to determine whether prostasin promotes the release of the intervening tract between the putative furin and γRKRK186 cleavage sites. Anti-γ43 precipitated both full-length (93 kDa) and furin-processed (83 kDa) γ-subunits from extracts obtained from oocytes expressing αßHA-γ-V5 channels, but only the full-length (93 kDa) γ-subunit from oocytes expressing αßHA-γ-V5 channels and either wild-type or a catalytically inactive prostasin. Although both wild-type and catalytically inactive prostasin activated ENaCs in an aprotinin-sensitive manner, only wild-type prostasin bound to aprotinin beads, suggesting that catalytically inactive prostasin facilitates the cleavage of the γ-subunit by an endogenous protease in Xenopus oocytes. As dietary salt restriction increases cleavage of the renal γ-subunit, we assessed release of the 43-mer inhibitory tract on rats fed a low-Na+ diet. We found that a low-Na+ diet increased γ-subunit cleavage detected with the anti-γ antibody and dramatically reduced the fraction precipitated with the anti-γ43 antibody. Our results suggest that the inhibitory tract dissociates from the γ-subunit in kidneys from rats on a low-Na+ diet.

Cysteine palmitoylation of the γ subunit has a dominant role in modulating activity of the epithelial sodium channel.

The epithelial sodium channel (ENaC) is composed of three homologous subunits (α, ß, and γ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the ß subunit, ßCys-43 and ßCys-557, are Cys-palmitoylated. ENaCs with mutant ßC43A/C557A exhibit normal surface expression but enhanced Na(+) self-inhibition and reduced channel open probability. Although the α subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the γ subunit are palmitoylated. ENaCs with mutant γC33A, γC41A, or γC33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of α and γ subunit-activating cleavage. These mutant channels have significantly enhanced Na(+) self-inhibition and reduced open probability compared with wild type ENaCs. Channel activity was enhanced by co-expression with the palmitoyltransferase DHHC2 that also co-immunoprecipitates with ENaCs. Secondary structure prediction of the N terminus of the γ subunit places γCys-33 within an α-helix and γCys-44 on a coil before the first transmembrane domain within a short tract that includes a well conserved His-Gly motif, where mutations have been associated with altered channel gating. Our current and previous results suggest that palmitoylation of the ß and γ subunits of ENaCs enhances interactions of their respective cytoplasmic domains with the plasma membrane and stabilizes the open state of the channel. Comparison of activities of channels lacking palmitoylation sites in individual or multiple subunits revealed that γ subunit palmitoylation has a dominant role over ß subunit palmitoylation in modulating ENaC gating.

Multiple residues in the distal C terminus of the α-subunit have roles in modulating human epithelial sodium channel activity.

Epithelial sodium channels (ENaC) are critically important in the regulation of ion and fluid balance in both renal and respiratory epithelia. ENaC functional polymorphisms may contribute to alterations in blood pressure in the general population. We previously reported that the A663T polymorphism in the C terminus of the α-subunit altered ENaC functional and surface expression in Xenopus laevis oocytes (Samaha FF, Rubenstein RC, Yan W, Ramkumar M, Levy DI, Ahn YJ, Sheng S, Kleyman TR. J Biol Chem 279: 23900-23907, 2004). We examined whether sites in the vicinity of 663 influenced channel activity by performing scanning Ala mutagenesis. Interestingly, only αT663/G667Aßγ channels exhibited increased currents compared with αT663ßγ. This increase in channel activity reflected an increase in channel open probability and not an increase in channel surface expression. In contrast, decreases in channel activity were observed with both αT663/C664Aßγ and αT663/C664Mßγ channels. The decrease in functional expression of αT663/C664Mßγ channels correlated with decreased surface expression, suggesting that the αC664M mutation altered the intracellular trafficking of the channel. While cytoplasmic Cys residues may be modified by the addition of palmitate, we did not observe palmitoylation of αC664. Our results suggest that multiple residues in the distal part of the cytoplasmic C terminus have roles in modulating channel activity.

TMPRSS4-dependent activation of the epithelial sodium channel requires cleavage of the γ-subunit distal to the furin cleavage site.

The epithelial sodium channel (ENaC) is activated by a unique mechanism, whereby inhibitory tracts are released by proteolytic cleavage within the extracellular loops of two of its three homologous subunits. While cleavage by furin within the biosynthetic pathway releases one inhibitory tract from the α-subunit and moderately activates the channel, full activation through release of a second inhibitory tract from the γ-subunit requires cleavage once by furin and then at a distal site by a second protease, such as prostasin, plasmin, or elastase. We now report that coexpression of mouse transmembrane protease serine 4 (TMPRSS4) with mouse ENaC in Xenopus oocytes was associated with a two- to threefold increase in channel activity and production of a unique ∼70-kDa carboxyl-terminal fragment of the γ-subunit, similar to the ∼70-kDa γ-subunit fragment that we previously observed with prostasin-dependent channel activation. TMPRSS4-dependent channel activation and production of the ∼70-kDa fragment were partially blocked by mutation of the prostasin-dependent cleavage site (γRKRK186QQQQ). Complete inhibition of TMPRSS4-dependent activation of ENaC and γ-subunit cleavage was observed when three basic residues between the furin and prostasin cleavage sites were mutated (γK173Q, γK175Q, and γR177Q), in addition to γRKRK186QQQQ. Mutation of the four basic residues associated with the furin cleavage site (γRKRR143QQQQ) also prevented TMPRSS4-dependent channel activation. We conclude that TMPRSS4 primarily activates ENaC by cleaving basic residues within the tract γK173-K186 distal to the furin cleavage site, thereby releasing a previously defined key inhibitory tract encompassing γR158-F168 from the γ-subunit.

Cys palmitoylation of the beta subunit modulates gating of the epithelial sodium channel.

The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (α, ß, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the ß and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant ß subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a ß C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na(+) self-inhibition, and reduced single channel P(o) when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that ß Cys-43 is in proximity to the first transmembrane α helix, whereas ß Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that ß subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.

Plasmin activates epithelial Na+ channels by cleaving the gamma subunit.

Proteolytic processing of epithelial sodium channel (ENaC) subunits occurs as channels mature within the biosynthetic pathway. The proteolytic processing events of the alpha and gamma subunits are associated with channel activation. Furin cleaves the alpha subunit ectodomain at two sites, releasing an inhibitory tract and activating the channel. However, furin cleaves the gamma subunit ectodomain only once. A second distal cleavage in the gamma 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 gamma subunit at gammaLys194, a site distal to the furin site. A gammaK194A mutant prevented both plasmin-dependent activation of ENaC and plasmin-dependent production of a unique 70-kDa carboxyl-terminal gamma 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.

Epithelial sodium channel exit from the endoplasmic reticulum is regulated by a signal within the carboxyl cytoplasmic domain of the alpha subunit.

Epithelial sodium channels (ENaCs) are assembled in the endoplasmic reticulum (ER) from alpha, beta, and gamma subunits, each with two transmembrane domains, a large extracellular loop, and cytoplasmic amino and carboxyl termini. ENaC maturation involves transit through the Golgi complex where Asn-linked glycans are processed to complex type and the channel is activated by furin-dependent cleavage of the alpha and gamma subunits. To identify signals in ENaC for ER retention/retrieval or ER exit/release, chimera were prepared with the interleukin alpha subunit (Tac) and each of the three cytoplasmic carboxyl termini of mouse ENaC (Tac-Ct) or with gamma-glutamyltranspeptidase and each of the three cytoplasmic amino termini (Nt-GGT). By monitoring acquisition of endoglycosidase H resistance after metabolic labeling, we found no evidence of ER retention of any chimera when compared with control Tac or GGT, but we did observe enhanced exit of Tac-alphaCt when compared with Tac. ER exit of ENaC was assayed after metabolic labeling by following the appearance of cleaved alpha as cleaved alpha subunit, but not non-cleaved alpha, is endoglycosidase H-resistant. Interestingly ER exit of epitope-tagged and truncated alpha (alphaDelta624-699-V5) with full-length betagamma was similar to wild type alpha (+betagamma), whereas ER exit of ENaC lacking the entire cytoplasmic carboxyl tail of alpha (alphaDelta613-699-V5 +betagamma) was significantly reduced. Subsequent analysis of ER exit for ENaCs with mutations within the intervening sequence (613)HRFRSRYWSPG(623) within the context of the full-length alpha revealed that mutation alphaRSRYW(620) to AAAAA significantly reduced ER exit. These data indicate that ER exit of ENaC is regulated by a signal within the alpha subunit carboxyl cytoplasmic tail.

Small heat shock protein alphaA-crystallin regulates epithelial sodium channel expression.

Integral membrane proteins are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). After being translocated or inserted into the ER, they fold and undergo post-translational modifications. Within the ER, proteins are also subjected to quality control checkpoints, during which misfolded proteins may be degraded by proteasomes via a process known as ER-associated degradation. Molecular chaperones, including the small heat shock protein alphaA-crystallin, have recently been shown to play a role in this process. We have now found that alphaA-crystallin is expressed in cultured mouse collecting duct cells, where apical Na(+) transport is mediated by epithelial Na(+) channels (ENaC). ENaC-mediated Na(+) currents in Xenopus oocytes were reduced by co-expression of alphaA-crystallin. This reduction in ENaC activity reflected a decrease in the number of channels expressed at the cell surface. Furthermore, we observed that the rate of ENaC delivery to the cell surface of Xenopus oocytes was significantly reduced by co-expression of alphaA-crystallin, whereas the rate of channel retrieval remained unchanged. We also observed that alphaA-crystallin and ENaC co-immunoprecipitate. These data are consistent with the hypothesis that small heat shock proteins recognize ENaC subunits at ER quality control checkpoints and can target ENaC subunits for ER-associated degradation.

Maturation of the epithelial Na+ channel involves proteolytic processing of the alpha- and gamma-subunits.

The epithelial Na+ channel (ENaC) is a tetramer of two alpha-, one beta-, and one gamma-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 alpha-(95 kDa), beta-(96 kDa), and gamma-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of alphabetagammaENaC subunits revealed a second band for each subunit (65 kDa for alpha, 110 kDa for beta, and 75 kDa for gamma) 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 [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed alpha- and gamma-subunits is also consistent with proteolytic cleavage. By using alpha- and gamma-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the alpha- and gamma-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for alpha and 18 kDa for gamma) 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.

Effect of injecting primary myoblasts versus putative muscle-derived stem cells on mass and force generation in mdx mice.

It is well established that the injection of normal myoblasts or of muscle-derived stem cells (MDSCs) into the muscle of dystrophin-deficient mdx mice results in the incorporation of a number of donor myoblasts into the host muscle. However, the effect of the injected exogenous cells on mdx muscle mass and functional capacity has not been evaluated. This study evaluates the mass and functional capacity of the extensor digitorum longus (EDL) muscles of adult, male mdx mice that received intramuscular injections of primary myoblasts or of MDSCs (isolated by a preplating technique; Qu, Z., Balkir, L., van Deutekom, J.C., Robbins, P.D., Pruchnic, R., and Huard, J., J. Cell Biol. 1998;142:1257-1267) derived from normal mice. Evaluations were made 9 weeks after cell transplantation. Uninjected mdx EDL muscles have a mass 50% greater than that of age-matched C57BL/10J (normal) EDL muscles. Injections of either primary myoblasts or MDSCs have no effect on the mass of mdx EDL muscles. EDL muscles of mdx mice generate 43% more absolute twitch tension and 43% less specific tetanic tension then do EDL muscles of C57BL/10J mice. However, the absolute tetanic and specific twitch tension of mdx and C57BL/10J EDL muscles are similar. Injection of either primary myoblasts or MDSCs has no effect on the absolute or specific twitch and tetanic tensions of mdx muscle. Approximately 25% of the myofibers in mdx EDL muscles that received primary myoblasts react positively with antibody to dystrophin. There is no significant difference in the number of dystrophin-positive myofibers when MDSCs are injected. Regardless of the source of donor cells, dystrophin is limited to short distances (60-900 microm) along the length of the myofibers. This may, in part, explain the failure of cellular therapy to alter the contractile properties of murine dystrophic muscle.