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Item does not contain fulltext Pathogenic mutations in TMPRSS3, which encodes a transmembrane serine protease, cause non-syndromic deafness DFNB8/10. Missense mutations map in the low density-lipoprotein receptor A (LDLRA), scavenger-receptor cysteine-rich (SRCR), and protease domains of the protein, indicating that all domains are important for its function. TMPRSS3 undergoes proteolytic cleavage and activates the ENaC sodium channel in a Xenopus oocyte model system. To assess the importance of this gene in non-syndromic childhood or congenital deafness in Turkey, we screened for mutations affected members of 25 unrelated Turkish families. The three families with the highest LOD score for linkage to chromosome 21q22.3 were shown to harbor P404L, R216L, or Q398X mutations, suggesting that mutations in TMPRSS3 are a considerable contributor to non-syndromic deafness in the Turkish population. The mutant TMPRSS3 harboring the novel R216L missense mutation within the predicted cleavage site of the protein fails to undergo proteolytic cleavage and is unable to activate ENaC, thus providing evidence that pre-cleavage of TMPRSS3 is mandatory for normal function.

CAP1/Prss8 is a membrane-bound serine protease involved in the regulation of several different effectors, such as the epithelial sodium channel ENaC, the protease-activated receptor PAR2, the tight junction proteins, and the profilaggrin polypeptide. Recently, the V170D and the G54-P57 deletion mutations within the CAP1/Prss8 gene, identified in mouse frizzy (fr) and rat hairless (fr(CR)) animals, respectively, have been proposed to be responsible for their skin phenotypes. In the present study, we analyzed those mutations, revealing a change in the protein structure, a modification of the glycosylation state, and an overall reduction in the activation of ENaC of the two mutant proteins. In vivo analyses demonstrated that both fr and fr(CR) mutant animals present analogous reduction of embryonic viability, similar histologic aberrations at the level of the skin, and a significant decrease in the activity of ENaC in the distal colon compared with their control littermates. Hairless rats additionally had dehydration defects in skin and intestine and significant reduction in the body weight. In conclusion, we provided molecular and functional evidence that CAP1/Prss8 mutations are accountable for the defects in fr and fr(CR) animals, and we furthermore demonstrate a decreased function of the CAP1/Prss8 mutant proteins. Therefore, fr and fr(CR) animals are suitable models to investigate the consequences of CAP1/Prss8 action on its target proteins in the whole organism.

Transepithelial sodium transport via alveolar epithelial Na(+) channels and Na(+),K(+)-ATPase constitutes the driving force for removal of alveolar oedema fluid. Decreased activity of the amiloride-sensitive epithelial Na(+) channel (ENaC) in the apical membrane of alveolar epithelial cells impairs sodium-driven alveolar fluid clearance (AFC) and predisposes to pulmonary oedema. We hypothesized that hyperactivity of ENaC in the distal lung could improve AFC and facilitate the resolution of pulmonary oedema. AFC and lung fluid balance were studied at baseline and under conditions of hydrostatic pulmonary oedema in the beta-Liddle (L) mouse strain harbouring a gain-of-function mutation (R(566)(stop)) within the Scnn1b gene. As compared with wild-type (+/+), baseline AFC was increased by 2- and 3-fold in heterozygous (+/L) and homozygous mutated (L/L) mice, respectively, mainly due to increased amiloride-sensitive AFC. The beta(2)-agonist terbutaline stimulated AFC in +/+ and +/L mice, but not in L/L mice. Acute volume overload induced by saline infusion (40% of body weight over 2 h) significantly increased extravascular (i.e. interstitial and alveolar) lung water as assessed by the bloodless wet-to-dry lung weight ratio in +/+ and L/L mice, as compared with baseline. However, the increase was significantly larger in +/+ than in L/L groups (P=0.01). Volume overload also increased the volume of the alveolar epithelial lining fluid in +/+ mice, indicating the presence of alveolar oedema, but not in L/L mice. Cardiac function as evaluated by echocardiography was comparable in both groups. These data show that constitutive ENaC activation improved sodium-driven AFC in the mouse lung, and attenuated the severity of hydrostatic pulmonary oedema.

Mouse channel activating proteases 1, 2, and 3 (mCAP1, mCAP2, and mCAP3) were described recently as regulators of the epithelial sodium channel (ENaC). The mCAP are membrane-bound serine proteases that are synthesized as inactive proenzymes. To mature into active proteases, they undergo intramolecular cleavage by auto- and/or heterocatalytic processing. Specific antibodies against each mCAP were developed to distinguish between proenzyme and active protease by Western blot analysis. Various point mutations were introduced in the catalytic or protein–protein interacting domains of mCAP and wild-type and mutant enzymes were expressed in the Xenopus oocyte expression system to test for ability to activate ENaC. In mCAP3, an intact catalytic triad was necessary for activation of ENaC but not for intramolecular cleavage of the protease. This suggests a heterocatalytic mechanism. Mutating the catalytic triad of mCAP2 not only abolished ENaC activation completely but also impeded cleavage of the protease. Processing of mCAP2 therefore seems to be autocatalytic. Furthermore, mutations in conserved residues of mCAP2 located in two protein–protein interacting domains significantly modulated ENaC activation. Surprisingly, mCAP1 catalytically inactive mutants were still able to fully activate ENaC, and no evidence of mCAP1 intramolecular cleavage was seen. The presence of an intact glycosylphosphatidylinositol anchor, however, was required. It is concluded that auto- and heterocatalytic requirements are specific for each CAP and that endogenous partners are a necessity for activation of ENaC by mCAP in the Xenopus oocyte expression system.

Aldosterone controls the final sodium reabsorption and potassium secretion in the kidney by regulating the activity of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN). ASDN consists of the last portion of the distal convoluted tubule (late DCT), the connecting tubule (CNT), and the collecting duct (CD) (i.e., the cortical CD [CCD] and the medullary CD [MCD]). It has been proposed that the control of sodium transport in the CCD is essential for achieving sodium and potassium balance. We have tested this hypothesis by inactivating the alpha subunit of ENaC in the CD but leaving ENaC expression in the late DCT and CNT intact. Under salt restriction or under aldosterone infusion, whole-cell voltage clamp of principal cells of CCD showed no detectable ENaC activity, whereas large amiloride-sensitive currents were observed in control littermates. The animals survive well and are able to maintain sodium and potassium balance, even when challenged by salt restriction, water deprivation, or potassium loading. We conclude that the expression of ENaC in the CD is not a prerequisite for achieving sodium and potassium balance in mice. This stresses the importance of more proximal nephron segments (late DCT/CNT) to achieve sodium and potassium balance.

The autosomal recessive form of type I pseudohypoaldosteronism (PHA-I) is an inherited salt-losing syndrome resulting from diminution-of-function mutations in the 3 subunits of the epithelial Na+ channel (ENaC). A PHA-I stop mutation (alpha(R508stop)) of the ENaC alpha subunit is predicted to lack the second transmembrane domain and the intracellular COOH-terminus, regions of the protein involved in pore function. Nonetheless, we observed a measurable Na+ current in Xenopus laevis oocytes that coexpress the beta and gamma subunits with the truncated alpha subunit. The mutant alpha was coassembled with beta and gamma subunits and was present at the cell surface at a lower density, consistent with the lower Na+ current seen in oocytes with the truncated alpha subunit. The single-channel Na+ conductance for the mutant channel was only slightly decreased, and the appearance of the macroscopic currents was delayed by 48 hours with respect to wild-type. Our data suggest novel roles for the alpha subunit in the assembly and targeting of an active channel to the cell surface, and suggest that channel pores consisting of only the beta and gamma subunits can provide significant residual activity. This activity may be sufficient to explain the absence of a severe pulmonary phenotype in patients with PHA-I.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high- Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK. Refereed/Peer-reviewed

Transepithelial alveolar sodium (Na+) transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) constitutes the driving force for removal of fluid from the alveolar space. To define the role of the β-ENaC subunit in vivo in the mature lung, we studied a previously established mouse strain harboring a disruption of the β-ENaC gene locus resulting in low levels of β-ENaC mRNA expression. Real-time RT-PCR experiments confirmed that β-ENaC mRNA levels were decreased by >90% in alveolar epithelial cells from homozygous mutant (m/m) mice. β-ENaC protein was undetected in lung homogenates from m/m mice by Western blotting, but α- and γ-ENaC proteins were increased by 83% and 45%, respectively, compared with wild-type (WT) mice. At baseline, Na+-driven alveolar fluid clearance (AFC) was significantly reduced by 32% in m/m mice. Amiloride at the concentration 1 mM inhibited AFC by 75% and 34% in WT and m/m mice, respectively, whereas a higher concentration (5 mM) induced a 75% inhibition of AFC in both groups. The β2-agonist terbutaline significantly increased AFC in WT but not in m/m mice. These results show that despite the compensatory increase in α- and γ-ENaC protein expression observed in mutant mouse lung, low expression of β-ENaC results in a moderate impairment of baseline AFC and in decreased AFC sensitivity to amiloride, suggesting a possible change in the stoichiometry of ENaC channels. Finally, adequate β-ENaC expression appears to be required for AFC stimulation by β2-agonists.

Aldosterone controls sodium balance by regulating an epithelial sodium channel (ENaC)-mediated sodium transport along the aldosterone-sensitive distal nephron, which expresses both mineralocorticoid (MR) and glucocorticoid receptors (GR). Mineralocorticoid specificity is ensured by 11beta-hydroxysteroid dehydrogenase type 2, which metabolizes cortisol or corticosterone into inactive metabolites that are unable to bind MR and/or GR. The fractional occupancy of MR and GR by aldosterone mediating the sodium transport response in the aldosterone-sensitive distal nephron cannot be studied in vivo. For answering this question, a novel mouse cortical collecting duct cell line (mCCD(cl1)), which expresses significant levels of MR and GR and a robust aldosterone sodium transport response, was used. Aldosterone elicited a biphasic response: Low doses (K(1/2) = approximately 0.5 nM) induced a transient and early increase of sodium transport (peaking at 3 h), whereas high doses (K(1/2) = approximately 90 nM) entailed an approximately threefold larger, long-lasting response. At 3 h, the corticosterone dose-response curve was shifted to the right compared with that of aldosterone by more than two log concentrations, an effect that was fully reverted in the presence of the 11beta-hydroxysteroid dehydrogenase type 2 inhibitor carbenoxolone. Low doses of dexamethasone (0.1 to 1 nM) failed to induce an early response, but high doses elicited a long-lasting response (K(1/2) = approximately 8 nM), similar to that observed for high aldosterone concentrations. Equilibrium binding assays showed that both aldosterone and corticosterone bind to a high-affinity, low-capacity site, whereas dexamethasone binds to one site. Within the physiologic range of aldosterone concentrations, sodium transport is predicted to be controlled by MR occupancy during circadian cycles and by MR and GR occupancy during salt restriction or acute stress.

In the principal cell of the renal collecting duct, vasopressin regulates the expression of a gene network responsible for sodium and water reabsorption through the regulation of the water channel and the epithelial sodium channel (ENaC). We have recently identified a novel vasopressin-induced transcript (VIT32) that encodes for a 142 amino acid vasopressin-induced protein (VIP32), which has no homology with any protein of known function. The Xenopus oocyte expression system revealed two functions: (i) when injected alone, VIT32 cRNA rapidly induces oocyte meiotic maturation through the activation of the maturation promoting factor, the amphibian homolog of the universal M phase trigger Cdc2/cyclin; and (ii) when co-injected with the ENaC, VIT32 cRNA selectively downregulates channel activity, but not channel cell surface expression. In the kidney principal cell, VIP32 may be involved in the downregulation of transepithelial sodium transport observed within a few hours after vasopressin treatment. VIP32 belongs to a novel gene family ubiquitously expressed in oocyte and somatic cells that may be involved in G to M transition and cell cycling.

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (INa) by a GPI-anchored serine protease (mouse channel–activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase INa 6–10-fold in the Xenopus oocyte expression system. INa and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I125-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases INa 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcPo). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances INa by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcPo. Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in INa (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on INa was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.

This study examines whether serine proteases can activate the amiloride-sensitive sodium channel (ENaC) in mammalian kidney epithelial cells. The transepithelial sodium transport assessed by amiloride-sensitive short-circuit current appears to be sensitive to aprotinin, a protease inhibitor in a mouse cortical collecting duct cell line (mpkCCD(c14)). This result indicated that serine proteases may be implicated in the regulation of ENaC-mediated sodium transport. Using degenerated oligonucleotides to a previously isolated serine protease from Xenopus, xCAP1 (channel activating protease), a novel full-length serine protease (mCAP1), has been isolated and characterized. RNA analysis showed a broad pattern of expression in tissues (kidney, lung, colon, and salivary glands) expressing ENaC. Reverse transcription-PCR experiments also showed that mCAP1 was abundantly expressed in proximal tubule cells and was also expressed in intact and cultured collecting duct cells. Coexpression of the Xenopus, rat, or human alpha-, beta-, and gamma-ENaC subunits in Xenopus oocytes also showed that mCAP1 induces a significant increase in ENaC-mediated current accompanied by a decrease of channel molecules at the cell surface. It is proposed that this novel mouse channel activating protease may act as a regulator of ENaC within the kidney.