The mechanisms by which proteases activate the epithelial sodium channel (ENaC) are not yet fully understood. We investigated the effect of extracellular proteases on rat ENaC heterologously expressed in Xenopus laevis oocytes. Application of trypsin increased ENaC whole-oocyte currents by about 8-fold without a concomitant increase in channel surface expression. The stimulatory effect of trypsin was preserved in oocytes expressing αγ-ENaC, but was abolished in oocytes expressing αβ-ENaC. Thus, the γ-subunit appears to be essential for channel activation by extracellular proteases. Site-directed mutagenesis of a putative prostasin cleavage site in the extracellular loop of the γ-subunit revealed that mutating the 181Lys residue to alanine (γK181A) increases ENaC baseline whole-oocyte currents, decreases channel surface expression, and largely reduces the stimulatory effect of extracellular proteases (trypsin, chymotrypsin and human neutrophil elastase). In single-channel recordings from outside-out patches we demonstrated that the γK181A mutation essentially abolishes the activation of near-silent channels by trypsin, while a stimulatory effect of trypsin on channel gating is preserved. This apparent dual effect of trypsin on channel gating and on the recruitment of near-silent channels was confirmed by experiments using the β518C mutant ENaC which can be converted to a channel with an open probability of nearly one by exposure to a sulfhydryl reagent. Interestingly, the γK181A mutation results in the spontaneous appearance of a 67 kDa fragment of the γ-subunit in the plasma membrane which can be prevented by a furin inhibitor and also occurs after channel activation by extracellular trypsin. This suggests that the mutation promotes channel cleavage and activation by endogenous proteases. This would lower the pool of near-silent channels and explain the constitutive activation and reduced responsiveness of the mutant channel to extracellular proteases. We conclude that the mutated site (K181A) affects a region in the γ-subunit of ENaC that is functionally important for the activation of near-silent channels by extracellular proteases.

Aldosterone-induced serum-and glucocorticoid-inducible kinase isoform 1 (SGK1) contributes to the regulation of the epithelial sodium channel (ENaC), the activity of which is critical for long term blood pressure control. Aldosterone-induced SGK1 is thought to enhance ENaC surface expression by phosphorylating Nedd4-2 and thereby preventing ENaC retrieval and degradation. In outside-out membrane patches of Xenopus laevis oocytes heterologously expressing ENaC, amiloride-sensitive ENaC currents were enhanced by phosphatase inhibitors and were dependent on cytosolic Mg 2؉ . This indicates that a kinase is involved in channel regulation. Indeed, recombinant constitutively active SGK1, included in the pipette solution, caused a sustained 2-to 3-fold increase of ENaC currents. Deletion of the C terminus of ␣ENaC largely reduced the stimulatory effect of SGK1, whereas stimulation by SGK1 did not require the presence of the C termini of the ␤-or ␥-subunits. Replacing the serine residue Ser 621 of the SGK1 consensus motif in the C terminus of the ␣-subunit by an alanine specifically abolished the stimulatory effect of SGK. Our findings indicate that SGK1 can stimulate ENaC activity independently of an inhibition of Nedd4-2-mediated channel retrieval. This defines a novel regulatory pathway likely to be relevant for aldosterone-induced stimulation of ENaC in vivo.The appropriate regulation of the epithelial sodium channel (ENaC) 1 in the kidney is critically important for the maintenance of body sodium balance and hence for long term regulation of arterial blood pressure (1). Indeed, two human genetic diseases provide direct evidence that molecular dysfunction of ENaC has severe effects on arterial blood pressure. Loss-offunction mutations of ENaC cause urinary sodium loss, hyperkalemia, and low blood pressure in patients with pseudohypoaldosteronism type 1 (2). In contrast, gain-of-function mutations of ENaC are found in patients with so-called Liddle's syndrome (pseudohyperaldosteronism) and result in increased renal sodium re-absorption, hypokalemia, and severe arterial hypertension (3).ENaC is composed of three subunits called ␣, ␤, and ␥ (4). The C termini of the ENaC subunits each contain a proline-rich PPXY (PY) motif, which is believed to be important for interaction with the ubiquitin-protein ligases Nedd4 and Nedd4-2, promoting the ubiquitination, endocytosis, and proteasomal degradation of the channel (5-8). The functional importance of the PY motif was recognized in Liddle's syndrome where mutations and/or deletions of the PY motif in ␤ or ␥ ENaC reduce the endocytic retrieval of ENaC from the membrane (9, 10). This results in an increase in the number of ENaC channels in the membrane, which in turn is thought to cause hyperabsorption of Na ϩ and hypertension in patients with Liddle's syndrome (11,12).The most important hormone to regulate ENaC activity is the mineralocorticoid aldosterone. The effects of aldosterone include transcriptional, translational, and post-translational modifications of ENaC and inv...

Increased activity of the epithelial sodium channel (ENaC) in the respiratory airways contributes to the pathophysiology of cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some patients suffering from atypical CF a mutation can be identified in only one CFTR allele. We recently identified in this group of CF patients a heterozygous mutation (W493R) in the α-subunit of ENaC. Here, we investigate the functional effects of this mutation by expressing wild-type αβγENaC or mutant α W493R βγENaC in Xenopus oocytes. The αW493R mutation stimulated amiloride-sensitive whole-cell currents ( I ami ) by ∼4-fold without altering the single-channel conductance or surface expression of ENaC. As these data suggest that the open probability (P o ) of the mutant channel is increased, we investigated the proteolytic activation of ENaC by chymotrypsin. Single-channel recordings revealed that chymotrypsin activated near-silent channels in outside-out membrane patches from oocytes expressing wild-type ENaC, but not in membrane patches from oocytes expressing the mutant channel. In addition, the αW493R mutation abolished Na + self inhibition of ENaC, which might also contribute to its gain-of-function effects. We conclude that the αW493R mutation promotes constitutive activation of ENaC by reducing the inhibitory effect of extracellular Na + and decreasing the pool of near-silent channels. The resulting gain-of-function phenotype of the mutant channel might contribute to the pathophysiology of CF in patients carrying this mutation.

Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its γ subunit in a critical region targeted by several proteases. Our aim was to identify cleavage sites in this region that are functionally important for activation of human ENaC by plasmin and chymotrypsin. Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) αβγENaC and αβγK189AENaC in Xenopus laevis oocytes. The γK189A mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (γRKRK178AAAA) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γRKRK178AAAA;K189A) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189, the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wt or double mutant ENaC. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near-silent channels was confirmed in single-channel recordings from outside-out patches. Mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. This indicates that chymotrypsin preferentially cleaves at FF174. The close proximity of FF174 to the prostasin site may explain why mutating the prostasin site impedes channel activation by chymotrypsin. In conclusion, this study supports the concept that different proteases have distinct preferences for certain cleavage sites in γENaC, which may be relevant for tissue-specific proteolytic ENaC activation.

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (␣, ␤, and ␥); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the ␣-, ␤-and ␥-subunits. We show that for ␣-, ␤-and ␥-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that ␣␤␥-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits.The epithelial sodium channel (ENaC) 5 is a member of the ENaC/degenerin superfamily, which also includes acid-sensing ion channels (ASICs; reviewed in Ref. 1). ENaC mediates Na ϩ entry across the apical membranes of many absorptive epithelia, including the alveolar epithelium, salivary duct, distal colon, and sweat glands. ENaC plays a particularly important role in Na ϩ transport across the aldosterone-sensitive distal nephron (reviewed in Ref. 2), which is responsible for the regulation of Na ϩ and K ϩ excretion, and thus plays an important role in the control of blood pressure. ENaC is the target of potassiumsparing diuretics such as amiloride and spironolactone used in the treatment of cardiovascular disease. Whereas amiloride is a direct blocker of the channel, the mineralocorticoid receptor antagonist spironolactone inhibits ENaC activity by preventing its stimulation by aldosterone.ENaC is a heteromultimer containing three homologous subunits (␣, ␤, and ␥), which have 30 -40% amino acid identity (3, 4). A fourth ENaC subunit, ␦-ENaC, has been cloned from a human kidney library (5). In heterologous expression systems, ␦-ENaC has functional similarities with ␣-ENaC, but its physiological role is still unclear (6). Each ENaC subunit contains two transmembrane domains, a large extracellular loop, and short intracellular N and C termini. Full ENaC activity requires coexpression of all three subunits (4). In the past, it has been proposed that ENaC is assembled from either four (7-9) or eight to nine subunits (10, 11). However, the recently published crystal structure of ASIC 1a (12) suggests that ENaC is likely to be a trimer (13), although this has not been demonstrated.We have developed a method, based on AFM imaging, for determining the stoichiometry and arrangement of subunits within multimeric proteins (reviewed in Ref. 14). The method involves engineering specific epitope tags onto each protein subunit and expressing the proteins exogenously in a suitable cell line (usually tsA 201). Membrane fractions from the transfected cells are solubilized in detergent, and the proteins are isola...

Several Cl(-) channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl(-) absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl(-)>Br(-)>NO3(-)>I(-). Single-channel recordings revealed a unit conductance of ~40pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~-65mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~20pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~+25mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253-260).

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin ion channel family, which also includes the bile acid-sensitive ion channel (BASIC). So far little is known about the effects of bile acids on ENaC function. ENaC is probably a heterotrimer consisting of three well characterized subunits (␣␤␥). In humans, but not in mice and rats, an additional ␦-subunit exists. The aim of this study was to investigate the effects of chenodeoxycholic, cholic, and deoxycholic acid in unconjugated (CDCA, CA, and DCA) and tauro-conjugated (t-CDCA, t-CA, t-DCA) form on human ENaC in its ␣␤␥-and ␦␤␥-configuration. We demonstrated that tauro-conjugated bile acids significantly stimulate ENaC in the ␣␤␥-and in the ␦␤␥-configuration. In contrast, non-conjugated bile acids have a robust stimulatory effect only on ␦␤␥ENaC. Bile acids stimulate ENaC-mediated currents by increasing the open probability of active channels without recruiting additional near-silent channels known to be activated by proteases. Stimulation of ENaC activity by bile acids is accompanied by a significant reduction of the single-channel current amplitude, indicating an interaction of bile acids with a region close to the channel pore. Analysis of the known ASIC1 (acid-sensing ion channel) crystal structure suggested that bile acids may bind to the pore region at the degenerin site of ENaC. Substitution of a single amino acid residue within the degenerin region of ␤ENaC (N521C or N521A) significantly reduced the stimulatory effect of bile acids on ENaC, suggesting that this site is critical for the functional interaction of bile acids with the channel.

The lipid environment of the epithelial sodium channel (ENaC) and its possible association with so-called lipid rafts may be relevant to its function. The aim of our study was to confirm the association of ENaC with lipid rafts and to analyze the effect of cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin (MβCD) on channel function and regulation. Using sucrose density gradient centrifugation we demonstrated that a significant portion of ENaC protein distributes to low density fractions thought to be typical lipid raft fractions. Importantly, cholesterol depletion of cell lysate by MβCD shifted ENaC to non-raft fractions of higher density. Live cell imaging demonstrated that treatment with MβCD largely reduced filipin staining over time, confirming cholesterol depletion of the plasma membrane. For electrophysiological studies intact oocytes were exposed to 20 mM MβCD for three hours. MβCD treatment had no consistent effect on baseline whole-cell ENaC currents. In addition to the typical single channel conductance of about 5 pS, subconductance states of ENaC were occasionally observed in patches from MβCD treated but not from control oocytes. Importantly, in outside-out patch clamp recordings the stimulatory effect of recombinant SGK1 in the pipette solution was essentially abolished in oocytes pretreated with MβCD. These results indicate that ENaC activation by cytosolic SGK1 is compromised by removing cholesterol from the plasma membrane. Thus, ENaC activation by SGK1 may require the presence of an intact lipid environment and/or of lipid rafts as signalling platform.