This study describes new yeast expression systems for each subunit of the heterotrimeric epithelial sodium channel (ENaC). We found that a significant amount of each subunit resides in the ER and is destroyed via ERAD. We also found that the chaperone requirements for ENaC subunit degradation were unlike any other ERAD substrate examined.

Activity of the epithelial Na؉ channel (ENaC) is modulated by Na ؉ self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na ؉ . We searched for determinants of Na ؉ self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at ␥Met 438 altered the Na ؉ self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na ؉ self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na ؉ self-inhibition. Mutations at the homologous site in the ␣ subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na ؉ self-inhibition. Mutations at the homologous ␤Ala 422 resulted in minimal or no change in Na ؉ self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing ␣G481M␤␥, ␣␤␥M438V, and ␣G481M/ ␤␥M438V, respectively. This pair of residues map to the ␣5 helix in the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both ␣G481C␤␥ and ␣␤␥M438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that ␣Gly 481 and ␥Met 438 are functional determinants of Na ؉ self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.Maintenance of body fluid volume homeostasis requires a collaborative interaction of many Na ϩ transport mechanisms. Na ϩ transport in epithelia that line the late distal convoluted tubule, connecting tubule, and collecting tubule relies on apical Na ϩ entry through epithelial Na ϩ channels (ENaC self-inhibition (4, 6 -8). However, detailed elements regarding its mechanism have not been revealed.A logical place to search for structural elements associated with Na ϩ self-inhibition is the large extracellular domain (ECD) that connects the two transmembrane domains (M1 and M2) within each ENaC subunit. The ECD likely exists as well structured subdomains with 16 conserved Cys residues. We recently reported that point mutations at multiple ␣ and ␥ ECD Cys residues blunted Na ϩ self-inhibition, and certain double or triple mutations rendered ENaC insensitive to high concentration of extracellular Na ϩ . These results suggest that multiple Cys residues are required to establish the proper tertiary structure permitting this allosteric regulation (9). In addition, the N-terminal portion of ECD contains ␥His 239 , a previously identified residue critical for Na ϩ self-inhibition, as well as defined protease cleavage sites (4, 10 -12). Various proteases have been shown to regulate ENaC activity, in part, by interfering with Na ϩ self-inhibition (6, 7, 13). The resolved high resolution structure of the chicken acidsensing i...

The epithelial Na ؉ channel (ENaC) mediates the rate-limiting step in transepithelial Na ؉ transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the ␣ subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the fingerthumb domain interface.The epithelial Na ϩ channel (ENaC) 2 is expressed at the apical surface of Na ϩ -transporting epithelia such as the distal nephron of the kidney, distal colon, and lung alveoli and airway. In conjunction with the Na ϩ /K ϩ -ATPase, ENaC transfers Na ϩ from the luminal to the interstitial space. This transfer is crucial in regulating blood pressure through its role in renal Na ϩ absorption and in regulating airway surface liquid volume and mucociliary clearance through its role in airway Na ϩ absorption. In accord with its role in these processes, improper ENaC function is implicated in several disorders. There is a growing body of evidence that enhanced ENaC activity in the airways of individuals with cystic fibrosis contributes to depletion of airway surface liquids resulting in poor mucociliary clearance (1-3). In the kidney, increased levels of aldosterone activate ENaC and increase the reabsorption of filtered Na ϩ (4). In both instances, increases in channel activity reflect, in part, enhanced channel proteolysis. Proteinuric states, characterized by excessive protein in the urine, are often accompanied by renal Na ϩ retention, volume expansion, and hypertension. Recent work indicates that volume expansion in proteinuric states reflects proteolytic activation of ENaC (5-7).ENaC is a trimer composed of three homologous subunits, ␣, ␤, and ␥ (8, 9). ENaC subunits are members of the much larger ENaC/Degenerin family of ion channel proteins. These channels share a few salient features as follows: 1) most are gated by ligands and/or mechanical forces; 2) they are Na ϩ -permeable and blocked by amiloride, a potassium-sparing diuretic; and 3) each subunit has two transmembrane helices (six transmembrane helices for the full channel), short intracellular N and C termini, and a large extracellular region comprised of sever...

Sodium retention is a major clinical feature of nephrotic syndrome. The mechanisms responsible for sodium retention in this setting have been a subject of debate for years. Excessive sodium retention occurs in some individuals with nephrotic syndrome in the absence of activation of the renin-angiotensin-aldosterone system, suggesting an intrinsic defect in sodium excretion by the kidney. Recent studies have provided new insights regarding mechanisms by which sodium transporters are activated by factors present in nephrotic urine. These mechanisms likely have a role in the development of hypertension in nephrotic syndrome, where hypertension may be difficult to control, and provide new therapeutic options for the management of blood pressure and edema in the setting of nephrotic syndrome.

Aldosterone is a key regulator of epithelial Na+ channels (ENaC) in renal cortical collecting ducts (CCD). The goal of this study was to examine whether serum- and glucocorticoid-inducible kinase-1 (SGK1), an aldosterone-induced gene, is vital to the delayed effect of aldosterone by increasing the gene expression of ENaC subunits. To test this hypothesis, we compared the levels of ENaC mRNA in mouse CCD cells that stably express either full-length (FL)-SGK1 or a kinase-dead dominant negative (K127M)-SGK1. Our results revealed that SGK1 regulates gene expression of ENaC, whether cells are maintained in steroid-free media or in the presence of corticosteroids (CS) and/or other growth factors. Under all conditions, the loss of function of SGK1 caused a significant decrease in the expression of alpha- and beta-ENaC, but not gamma-ENaC. Compared with cells expressing FL-SGK1, K127M-SGK1 decreased the expression of alpha- and beta-subunit mRNA by approximately 45 and approximately 90%, respectively. Next, to determine whether SGK1 is one of the proteins mediating the induction of alpha-ENaC mRNA by CS, we compared steroid induction of alpha-ENaC in cells expressing K127M-SGK1 vs. FL-SGK1. The maximum level of alpha-ENaC mRNA levels following CS was significantly (approximately 45%) higher in FL-SGK1- vs. K127M-SGK1-expressing cells, although the fold-induction by CS was similar in both FL-SGK1- and K127M-SGK1-expressing cells. In summary, we report for the first time that SGK1 regulates transcription of ENaC subunits. We propose that the effect of SGK1 on ENaC transcription is mediated by the activation of unidentified transcription factors.