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Cystic fibrosis (CF) remains the most lethal genetic disease in the Caucasian population. However, there is great variability in clinical phenotypes and survival times, even among patients harboring the same genotype. We identified five patients with CF and a homozygous F508del mutation in the CFTR gene who were in their fifth or sixth decade of life and had shown minimal changes in lung function over a longitudinal period of more than 20 years. Because of the rarity of this long-term nonprogressive phenotype, we hypothesized these individuals may carry rare genetic variants in modifier genes that ameliorate disease severity. Individuals at the extremes of survival time and lung-function trajectory underwent whole-exome sequencing, and the sequencing data were filtered to include rare missense, stopgain, indel, and splicing variants present with a mean allele frequency of

Epithelial Na+ channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (αF2M mice). On a normal Na+ control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild‐type (WT) and αF2M mice. Patch‐clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male αF2M mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride‐sensitive short‐circuit current (ISC). Following dietary Na+ restriction, WT and αF2M mice had similar natriuretic and colonic ISC responses to amiloride. However, single‐channel activity was significantly lower in kidney tubules from Na+‐restricted αF2M mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na+‐restricted αF2Mvs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na+ restriction. Key points: The epithelial Na+ channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole‐animal physiology remain to be addressed.We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC's α subunit (αF2M mice).We found that αF2M mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction.ENaC function at the organ level was preserved in salt‐restricted αF2M mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis.These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo. [ABSTRACT FROM AUTHOR]

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo. NEW & NOTEWORTHY: The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets. [ABSTRACT FROM AUTHOR]

Background: The epithelial Na+ channel (ENaC) is intrinsically linked to fluid volume homeostasis and blood pressure. Specific rare mutations in SCNN1A, SCNN1B, and SCNN1G, genes encoding the α, β, and γ subunits of ENaC, respectively, are associated with extreme blood pressure phenotypes. No associations between blood pressure and SCNN1D, which encodes the δ subunit of ENaC, have been reported. A small number of sequence variants in ENaC subunits have been reported to affect functional transport in vitro or blood pressure. The effects of the vast majority of rare and low-frequency ENaC variants on blood pressure are not known.Methods: We explored the association of low frequency and rare variants in the genes encoding ENaC subunits, with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure. Using whole-genome sequencing data from 14 studies participating in the Trans-Omics in Precision Medicine Whole-Genome Sequencing Program, and sequence kernel association tests.Results: We found that variants in SCNN1A and SCNN1B were associated with diastolic blood pressure and mean arterial pressure (P<0.00625). Although SCNN1D is poorly expressed in human kidney tissue, SCNN1D variants were associated with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure (P<0.00625). ENaC variants in 2 of the 4 subunits (SCNN1B and SCNN1D) were also associated with estimated glomerular filtration rate (P<0.00625), but not with stroke.Conclusions: Our results suggest that variants in extrarenal ENaCs, in addition to ENaCs expressed in kidneys, influence blood pressure and kidney function. [ABSTRACT FROM AUTHOR]

Epithelial [Na.sup.+] channels (ENaC) are inhibited by extracellular [Na.sup.+], a process referred to as [Na.sup.+] self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in [alpha], or one site in [gamma] blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary cells. We now report that channels with mutant [alpha]-subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the [Na.sup.+] self-inhibition response, while channels with a mutant [gamma]/-subunit showed a modestly enhanced [Na.sup.+] self-inhibition response. Analysis of [Na.sup.+] self-inhibition at varying [Na.sup.+] indicates that channels containing mutant [alpha]-subunits exhibit an increased [Na.sup.+] affinity. At the single-channel level, channels with a mutant [alpha]-subunit had a low open probability ([P.sub.o]) in the presence of a high external [Na.sup.+] in the patch pipette. [P.sub.o] dramatically increased when trypsin was also present, or when a low external [Na.sup.+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving [Na.sup.+] self-inhibition and that activation requires that the [alpha]-subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC [P.sub.o] and extracellular [Na.sup.+], supporting the notion that [Na.sup.+] self-inhibition reflects a [P.sub.o] reduction due to high extracellular [Na.sup.+]. amiloride; open probability; voltage clamp; Xenopus laevis oocyte; mutagenesis

Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC [alpha]-subunit that confer a dominant negative phenotype on functional expression of [alpha][beta][gamma]-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse [alpha][beta][gamma]-ENaC with either 1) the [alpha]-subunit first membrane-spanning domain and short downstream hydrophobic domain ([alpha]-M1H1); 2) [alpha]-M1H1 and its downstream hydrophilic extracellular loop ([alpha]-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; [gamma]-GT) fused to the [alpha]-ECL ([gamma]-GT[alpha]-ECL); 4) the extracellular domain of a control type 1 trans-membrane protein (Tac) fused to the [alpha]-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-[alpha]-H2M2); or 5) the [alpha]-subunit cytoplasmic COOH terminus ([alpha]-Ct) significantly reduced amiloride-sensitive [Na.sup.+] currents in X. laevis oocytes. Functional expression of [Na.sup.+] channels was not inhibited when full-length [alpha][beta][gamma]-ENaC was coexpressed with either 1) the [alpha]-ECL lacking a signal-anchor sequence, 2) [alpha]-M1H1 and [alpha]-Ct expressed as a fusion protein, 3) full-length [gamma]-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either [alpha]-M1H1-ECL or [alpha]-Ct. Full-length FLAG-tagged [alpha], [beta], or [gamma]-ENaC coimmunoprecipitated with myc-tagged [alpha]-M1H1-ECL, whereas wild-type [gamma]-GT did not. These data suggest that multiple sites within the [alpha]-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex. amiloride-sensitive sodium channel; dominant negative mutants