Alexei Diakov

Cleavage in the γ‐subunit of the epithelial sodium channel (ENaC) plays an important role in the proteolytic activation of near‐silent channels

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.

A mutation of the epithelial sodium channel associated with atypical cystic fibrosis increases channel open probability and reduces Na+ self inhibition

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 αW493RβγENaC in Xenopus oocytes. The αW493R mutation stimulated amiloride‐sensitive whole‐cell currents (ΔIami) by ∼4‐fold without altering the single‐channel conductance or surface expression of ENaC. As these data suggest that the open probability (Po) 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.

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (α, β, and γ); 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 α-, β- and γ-subunits. We show that for α-, β- and γ-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 αβγ-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.

Cholesterol Depletion of the Plasma Membrane Prevents Activation of the Epithelial Sodium Channel (ENaC) by SGK1

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.

Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the γ subunit of the human epithelial sodium channel

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.

Characterization of the mouse ClC-K1/Barttin chloride channel

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).

Protein Kinase B Alpha (PKBα) Stimulates the Epithelial Sodium Channel (ENaC) Heterologously Expressed in Xenopus laevis Oocytes by Two Distinct Mechanisms

Kinases contribute to the regulation of the epithelial sodium channel (ENaC) in a complex manner. For example, SGK1 (serum- and glucocorticoid-inducible kinase type 1) enhances ENaC surface expression by phosphorylating Nedd4-2, thereby preventing ENaC retrieval and degradation. An additional mechanism of ENaC activation by SGK1 involves an SGK consensus motif (616RSRYWS621) in the C-terminus of the channel’s Α-subunit. This consensus motif may also be a target for ENaC regulation by protein kinase B Α (PKBΑ) known to be activated by insulin and growth factors. Therefore, we investigated a possible role of PKBΑ in the regulation of rat ENaC heterologously expressed in Xenopus laevis oocytes. We found that recombinant PKBΑ included in the pipette solution increased ENaC currents in outside-out patches by about 4-fold within 15-20 min. Replacing the serine residue S621 of the SGK consensus motif by an alanine (S621A) abolished this stimulatory effect. In co-expression experiments active PKBΑ but not catalytically inactive PKBΑ significantly increased ENaC whole-cell currents and surface expression by more than 50 % within 24 hours of co-expression. Interestingly, this stimulatory effect was preserved in oocytes expressing ENaC with the S621A mutation. We conclude that the acute stimulatory effect of PKBΑ involves a specific kinase consensus motif in the C-terminus of the channel’s Α-subunit. In contrast, the increase in channel surface expression caused by co-expression of PKBΑ does not depend on this site in the channel and is probably mediated by an effect on channel trafficking.

Pharmacological and electrophysiological characterization of the human bile acid-sensitive ion channel (hBASIC)

The human bile acid-sensitive ion channel (hBASIC) is a cation channel of the degenerin/epithelial Na+ channel gene family that is expressed in the intestinal tract and can be activated by bile acids. Here, we show that in addition to its sensitivity for bile acids, hBASIC shares further key features with its rat ortholog: it is blocked by extracellular divalent cations, is inhibited by micromolar concentrations of the diarylamidine diminazene, and activated by millimolar concentrations of flufenamic acid. Furthermore, we demonstrate that two major bile acids present in human bile, chenodeoxycholic acid and deoxycholic acid, activate hBASIC in a synergistic manner. In addition, we determined the single-channel properties of hBASIC in outside-out patch clamp recordings, revealing a single-channel conductance of about 11 pS and a high Na+ selectivity. Deoxycholic acid activates hBASIC in patch clamp recordings mainly by reducing the single-channel closed time. In summary, we provide a thorough functional characterization of hBASIC.

A mutation in the β-subunit of ENaC identified in a patient with cystic fibrosis-like symptoms has a gain-of-function effect.

In some patients with atypical cystic fibrosis (CF), only one allele of the CF transmembrane conductance regulator (CFTR) gene is affected. Mutations of the epithelial sodium channel (ENaC) may contribute to the pathophysiology of the disease in these patients. To functionally characterize a mutation in the β-subunit of ENaC (βV348M) recently identified in a patient with severe CF-like symptoms (Mutesa et al. 2009), we expressed wild-type (wt) αβγENaC or mutant αβV348MγENaC in Xenopus laevis oocytes. The βV348M mutation stimulated amiloride-sensitive whole-cell current (ΔI(ami)) by ∼40% but had no effect on surface expression or single-channel conductance of ENaC. Instead the mutation increased channel open probability (P(o)). Proteolytic activation of mutant ENaC by chymotrypsin was reduced compared with that of wt ENaC (∼3.0-fold vs. ∼4.2-fold), which is consistent with the increased baseline P(o) of mutant ENaC. Similarly, the ENaC activator S3969 stimulated mutant ENaC currents to a lesser degree (by ∼2.6-fold) than wt ENaC currents (by ∼3.5-fold). The gain-of-function effect of the βV348M mutation was confirmed by whole-cell current measurements in HEK293 cells transiently transfected with wt or mutant ENaC. Computational channel modeling in combination with functional expression of different βV348 mutants in oocytes suggests that the βV348M mutation increases channel P(o) by destabilizing the closed channel state. Our findings indicate that the gain-of-function effect of the βV348M mutation may contribute to CF pathophysiology by inappropriately increasing sodium and fluid absorption in the respiratory tract.

Functional characterization of a partial loss-of-function mutation of the epithelial sodium channel (ENaC) associated with atypical cystic fibrosis.

Loss-of-function mutations of the epithelial sodium channel (ENaC) may contribute to pulmonary symptoms resembling those of patients with atypical cystic fibrosis (CF). Recently, we identified a loss-of-function mutation in the α-subunit of ENaC (αF61L) in an atypical CF patient without mutations in CFTR. To investigate the functional effect of this mutation, we expressed human wild-type αβγ-ENaC or mutant α F61Lβγ-ENaC in Xenopus laevis oocytes. The αF61L mutation reduced the ENaC mediated whole-cell currents by ñ90%. In contrast, the mutation decreased channel surface expression only by ñ40% and did not alter the single-channel conductance. These findings indicate that the major effect of the mutation is a reduction of the average channel open probability (Po). This was confirmed by experiments using the βS520C mutant ENaC which can be converted to a channel with a Po of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. These experiments revealed that the mutation reduced the average Po of ENaC by ñ75%. Na+ self inhibition of the mutant channel was significantly enhanced, but the observed effect was too small to account for the large reduction in average channel Po. The ENaC-activator S3969 partially rescued the loss-of-function phenotype of the αF61L mutation. We conclude that the αF61L mutation may contribute to respiratory symptoms in atypical CF patients.