Bile acids potentiate proton‐activated currents in Xenopus laevis oocytes expressing human acid‐sensing ion channel (ASIC1a)

Acid‐sensing ion channels (ASICs) are nonvoltage‐gated sodium channels transiently activated by extracellular protons and belong to the epithelial sodium channel (ENaC)/Degenerin (DEG) family of ion channels. Bile acids have been shown to activate two members of this family, the bile acid‐sensitive ion channel (BASIC) and ENaC. To investigate whether bile acids also modulate ASIC function, human ASIC1a was heterologously expressed in Xenopus laevis oocytes. Exposing oocytes to tauro‐conjugated cholic (t‐CA), deoxycholic (t‐DCA), and chenodeoxycholic (t‐CDCA) acid at pH 7.4 did not activate ASIC1a‐mediated whole‐cell currents. However, in ASIC1a expressing oocytes the whole‐cell currents elicited by pH 5.5 were significantly increased in the presence of these bile acids. Single‐channel recordings in outside‐out patches confirmed that t‐DCA enhanced the stimulatory effect of pH 5.5 on ASIC1a channel activity. Interestingly, t‐DCA reduced single‐channel current amplitude by ~15% which suggests an interaction of t‐DCA with a region close to the channel pore. Molecular docking predicted binding of bile acids to the pore region near the degenerin site (G433) in the open conformation of the channel. Site‐directed mutagenesis demonstrated that the amino acid residue G433 is critically involved in the potentiating effect of bile acids on ASIC1a activation by protons

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 -subunit. This consensus motif may also be a target for ENaC regulation by protein kinase B (PKB) known to be activated by insulin and growth factors. Therefore, we investigated a possible role of PKBin the regulation of rat ENaC heterologously expressed in Xenopus laevis oocytes. We found that recombinant PKBincluded in the pipette solution increased ENaC currents in outsideout 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 PKBbut not catalytically inactive PKBsignificantly increased ENaC whole-cell currents and surface expression by more than 50 % within 24 hours of coexpression. Interestingly, this stimulatory effect was preserved in oocytes expressing ENaC with the S621A mutation. We conclude that the acute stimulatory effect of PKBinvolves a specific kinase consensus motif in the C-terminus of the channel’s -subunit. In contrast, the increase in channel surface expression caused by co-expression of PKBdoes not depend on this site in the channel and is probably mediated by an effect on channel trafficking

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-- cyclodextrin (MCD) 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 MCD shifted ENaC to non-raft fractions of higher density. Live cell imaging demonstrated that treatment with MCD largely reduced filipin staining over time, confirming cholesterol depletion of the plasma membrane. For electrophysiological studies intact oocytes were exposed to 20 mM MCD for three hours. MCD 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 MCD 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 MCD. 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

Transmembrane serine protease 2 (TMPRSS2) proteolytically activates the epithelial sodium channel (ENaC) by cleaving the channel’s γ-subunit

The epithelial sodium channel (ENaC) is a heterotrimer consisting of α-, β- and γ-subunits. Channel activation requires proteolytic release of inhibitory tracts from the extracellular domains of α- and γ-ENaC; however, the proteases involved in the removal of the γ-inhibitory tract remain unclear. In several epithelial tissues, ENaC is coexpressed with the transmembrane serine protease 2 (TMPRSS2). Here, we explored the effect of human TMPRSS2 on human αβγ-ENaC heterologously expressed in Xenopus laevis oocytes. We found coexpression of TMPRSS2 stimulated ENaC-mediated whole-cell currents by ∼3-fold, likely due to an increase in average channel open probability. Furthermore, TMPRSS2-dependent ENaC stimulation was not observed using a catalytically-inactive TMPRSS2 mutant and was associated with fully-cleaved γ-ENaC in the intracellular and cell surface protein fractions. This stimulatory effect of TMPRSS2 on ENaC was partially preserved when inhibiting its proteolytic activity at the cell surface using aprotinin, but was abolished when the γ-inhibitory tract remained attached to its binding site following introduction of two cysteine residues (S155C – Q426C) to form a disulfide bridge. In addition, computer simulations and site-directed mutagenesis experiments indicated TMPRSS2 can cleave γ-ENaC at sites both proximal and distal to the γ-inhibitory tract. This suggests a dual role of TMPRSS2 in the proteolytic release of the γ-inhibitory tract. Finally, we demonstrated that TMPRSS2-knockdown in cultured human airway epithelial cells (H441) reduced baseline proteolytic activation of endogenously expressed ENaC. Thus, we conclude TMPRSS2 is likely to contribute to proteolytic ENaC activation in epithelial tissues in vivo