The epithelial sodium channel (ENaC) is a heterotrimer typically composed of three homologous subunits (α, β, and γ). Humans and several other species express an additional δ-subunit that can replace α-ENaC in heterologous expression systems, thereby modifying channel properties. A unique feature of ENaC is its proteolytic activation. Proteases remove autoinhibitory tracts from the Gating Relief of Inhibition by Proteolysis (GRIP) domains of α- and γ-ENaC. Recent ENaC structural data revealed that these tracts occupy specific binding pockets within their respective subunits. Using molecular dynamics simulations, site-directed mutagenesis, and electrophysiological recordings, we identified a cluster of four functionally important aromatic residues within the binding pocket of the γ-autoinhibitory tract in human αβγ-ENaC. These residues were also essential for ENaC inhibition by the synthetic γ-11 peptide, corresponding to the key portion of the γ-inhibitory tract. The aromatic cluster was conserved in α-ENaC, where it mediated the inhibitory effect of the synthetic α-8 peptide. Interestingly, structurally similar GRIP domains were also identified in δ- and β-ENaC. Our data indicate that the GRIP domains of both β- and δ-ENaC lack corresponding autoregulatory tracts. Moreover, we show that proteolytic activation of human δβγ-ENaC results from cleavage of γ-ENaC, but not δ-ENaC, consistent with the absence of autoregulatory activity of the δ-GRIP domain. In summary, this study demonstrates that the GRIP domains play distinct functional roles across different ENaC subunits. Characterization of the inhibitory sites in γ- and α-ENaC may facilitate the development of novel peptidomimetic ENaC inhibitors with potential (patho)physiological implications.

Mutations in polycystin-1 (PC1) or polycystin-2 (PC2) cause autosomal-dominant polycystic kidney disease (ADPKD). Structural data suggest that one PC1 and three PC2 form heterotetrameric ion channels with an ion permeation pathway blocked by PC1 (R4100, R4107, and H4111) and PC2 (L677, N681) residues. Here, we demonstrate that replacing these residues with alanines results in a gain-of-function (GOF) PC2/PC1 construct with distinct selectivity properties compared to PC2 homomers. We also show preferential formation of PC2/PC1 heteromeric complexes over PC2 homomers. Re-interpretation of published PC2/PC1 cryo-electron microscopy data, combined with cysteine modification experiments, suggests that the pore-forming domain of PC1 adopts a canonical TRP channel-like conformation. This novel PC2/PC1 GOF construct offers the opportunity to investigate the functional impact of ADPKD mutations.

The epithelial sodium channel (ENaC) is crucial for sodium absorption in several epithelial tissues including lung and kidney. Its involvement in various renal and pulmonary disorders makes ENaC a potential drug target. High-throughput screening using the automated patch-clamp (APC) technique appears to be a promising approach to discover novel ENaC modulators with (patho-)physiological and therapeutic implications. The aim of this methodological study was to establish APC measurements of ENaC-mediated currents. First, we confirmed functional expression of ENaC in a HEK293 cell line stably transfected with human αβγ-ENaC using conventional manual whole-cell patch-clamp recordings. For APC measurements, a standard enzymatic cell-detachment procedure was used to prepare single cell suspensions. This resulted in a high success rate of APC recordings with amiloride inhibitable ENaC currents. Using a γ-inhibitory peptide and the small molecule ENaC activator S3969, we demonstrate that APC recordings could reveal inhibitory as well as stimulatory effects on ENaC. Interestingly, the enzymatic cell-detachment protocol resulted in partial proteolytic ENaC activation. The portion of proteolytically activated channels could be reduced by prolonged incubation of suspended cells in cell culture medium. This recovery protocol enhanced the relative stimulatory effect of chymotrypsin, a prototypical serine protease known to cause proteolytic ENaC activation. Thus, this protocol may be particularly useful for identifying novel ENaC activators mimicking proteolytic channel activation. In conclusion, we have established a high-throughput screening method for the identification of novel ENaC activators and inhibitors using APC.

Polycystin-2 (PC2) mutations are responsible for ∼15% of cases of autosomal-dominant polycystic kidney disease (ADPKD). PC2 belongs to the transient receptor potential ion channel family and can function as a homotetrameric nonselective cation channel. Little is known about its gating mechanism, and no specific PC2 activators or inhibitors have been identified. In this study, we performed a comparative electrophysiological analysis of two well-established gain-of-function PC2 constructs, PC2 F604P and PC2 L677A N681A, expressed in Xenopus laevis oocytes. Interestingly, PC2 F604P, but not PC2 L677A N681A, could be inhibited by several membrane-permeable cysteine-reactive compounds. In contrast, positively charged cysteine-reactive compounds had no inhibitory effect. Thus, the inhibitory effect probably involves covalent modification of intracellular cysteine residues. Consistent with this, site-directed mutagenesis revealed a critical functional role of the free cysteine residue C593, localized in the intracellular S4-S5 linker of PC2 F604P. Moreover, the ion channel function of PC2 F604P was disturbed by S4-S5 linker mutations flanking C593. Interestingly, several structurally unrelated amphiphilic substances mimicked the inhibitory effect of membrane-permeable cysteine-reactive compounds on PC2 F604P without affecting PC2 L677A N681A. Collectively, our data suggest a critical role of the S4-S5 linker and the plasma membrane lipid environment in F604P-triggered PC2 gating. Finally, we demonstrated that the ADPKD-associated mutation located within the S4-S5 linker (N580K) completely abolished or significantly reduced currents mediated by PC2 F604P or PC2 L677A N681A, respectively. These findings provide new insights into molecular mechanisms involved in PC2 gating.

Serum and glucocorticoid inducible kinase 1 (SGK1) is a key regulator of the epithelial sodium channel (ENaC). In rat ENaC, the serine residue 621 (S621) in the channel's α-subunit is essential for acute channel activation by SGK1 in outside-out patches. Phosphorylation at S621 probably turns previously silent channels into channels with a high open probability. This is reminiscent of proteolytic ENaC activation resulting from cleavage of the channel's γ-subunit at specific proximal and distal cleavage sites and the release of an inhibitory peptide tract. The first aim of this study was to demonstrate that human ENaC could also be activated acutely by SGK1 and that this depended on the homologous phosphorylation site S594 in human αENaC. Secondly, we wanted to explore whether human ENaC activation by SGK1 depended on the cleavage state of γENaC. Outside-out patch-clamp recordings in Xenopus laevis oocytes expressing human αβγENaC revealed the critical importance of S594 for acute channel activation by SGK1. The latter was not additive to proteolytic channel activation. Interestingly, preventing proximal cleavage in human γENaC completely abolished the stimulatory effect of SGK1. Moreover, tethering the inhibitory peptide in γENaC to its binding site via an engineered disulfide bond prevented stimulation by SGK1. We conclude that ENaC activation by SGK1 requires prior cleavage of γENaC at its proximal cleavage site. Together, these results reveal that SGK1-mediated stimulation of human ENaC is intricately linked to the proteolytic processing of the channel's γ-subunit, emphasizing a previously underappreciated interplay between kinase and protease regulatory pathways.

The epithelial sodium channel (ENaC) is essential for mediating sodium absorption in several epithelia. Its impaired function leads to severe disorders, including pseudohypoaldosteronism type 1 and respiratory distress. Therefore, pharmacological ENaC activators have potential therapeutic implications. Previously, a small molecule ENaC activator (S3969) was developed. So far, little is known about molecular mechanisms involved in S3969-mediated ENaC stimulation. Here, we identified an S3969 binding site in human ENaC by combining structure-based simulations with molecular biological methods and electrophysiological measurements of ENaC heterologously expressed in Xenopus laevis oocytes. We confirmed a previous observation that the extracellular loop of β-ENaC is essential for ENaC stimulation by S3969. Molecular dynamics (MD) simulations predicted critical residues in the thumb domain of β-ENaC (Arg388, Phe391, and Tyr406) that coordinate S3969 within a binding site localized at the β-γ-subunit interface. Importantly, mutating each of these residues reduced (R388H; R388A) or nearly abolished (F391G; Y406A) the S3969-mediated ENaC activation. MD simulations also suggested that S3969-mediated ENaC stimulation involved a movement of the α5 helix of the thumb domain of β-ENaC away from the palm domain of γ-ENaC. Consistent with this, the introduction of two cysteine residues (βR437C – γS298C) to form a disulfide bridge connecting these two domains prevented ENaC stimulation by S3969 unless the disulfide bond was reduced by DTT. Finally, we demonstrated that S3969 stimulated ENaC endogenously expressed in cultured human airway epithelial cells (H441). These new findings may lead to novel (patho-)physiological and therapeutic concepts for disorders associated with altered ENaC function.

Polycystin-2 (PC2) is mutated in ∼15% of patients with autosomal dominant polycystic kidney disease (ADPKD). PC2 belongs to the family of transient receptor potential (TRP) channels and can function as a homotetramer. We investigated whether three disease-associated mutations (F629S, C632R, or R638C) localized in the channel's pore loop alter ion channel properties of human PC2 expressed in Xenopus laevis oocytes. Expression of wild-type (WT) PC2 typically resulted in small but measurable Na+ inward currents in the absence of extracellular divalent cations. These currents were no longer observed when individual pore mutations were introduced in WT PC2. Similarly, Na+ inward currents mediated by the F604P gain-of-function (GOF) PC2 construct (PC2 F604P) were abolished by each of the three pore mutations. In contrast, when the mutations were introduced in another GOF construct, PC2 L677A N681A, only C632R had a complete loss-of-function effect, whereas significant residual Na+ inward currents were observed with F629S (∼15%) and R638C (∼30%). Importantly, the R638C mutation also abolished the Ca2+ permeability of PC2 L677A N681A and altered its monovalent cation selectivity. To elucidate the molecular mechanisms by which the R638C mutation affects channel function, molecular dynamics (MD) simulations were used in combination with functional experiments and site-directed mutagenesis. Our findings suggest that R638C stabilizes ionic interactions between Na+ ions and the selectivity filter residue D643. This probably explains the reduced monovalent cation conductance of the mutant channel. In summary, our data support the concept that altered ion channel properties of PC2 contribute to the pathogenesis of ADPKD.

Key PointsProteolytic activation of the epithelial sodium channel (ENaC) was compromised by transmembrane serine protease 2 deficiency in murine cortical collecting duct cells and native mouse kidney.To compensate for impaired ENaC activation, rise in plasma aldosterone in response to low-salt diet was enhanced in -/- mice.Tmprss2Transmembrane serine protease 2 may be a potential drug target to limit proteolytic ENaC activation in disorders with increased renal ENaC activity.BackgroundThe renal epithelial sodium channel (ENaC) is essential for sodium balance and BP control. ENaC undergoes complex proteolytic activation by not yet clearly identified tubular proteases. Here, we examined a potential role of transmembrane serine protease 2 (TMPRSS2).MethodsMurine ENaC and TMPRSS2 were (co)expressed in Xenopus laevis oocytes. ENaC cleavage and function were studied in TMPRSS2-deficient murine cortical collecting duct (mCCDcl1) cells and TMPRSS2-knockout (Tmprss2-/-) mice. Short-circuit currents (ISC) were measured to assess ENaC-mediated transepithelial sodium transport of mCCDcl1 cells. The mCCDcl1 cell transcriptome was studied using RNA sequencing. The effect of low-sodium diet with or without high potassium were compared in Tmprss2-/- and wild-type mice using metabolic cages. ENaC-mediated whole-cell currents were recorded from microdissected tubules of Tmprss2-/- and wild-type mice.ResultsIn oocytes, coexpression of murine TMPRSS2 and ENaC resulted in fully cleaved gamma-ENaC and approximately two-fold stimulation of ENaC currents. High baseline expression of TMPRSS2 was detected in mCCDcl1 cells without a stimulatory effect of aldosterone on its function or transcription. TMPRSS2 knockout in mCCDcl1 cells compromised gamma-ENaC cleavage and reduced baseline and aldosterone-stimulated ISC, which could be rescued by chymotrypsin. A compensatory transcriptional upregulation of other proteases was not observed. Tmprss2-/- mice kept on standard diet exhibited no apparent phenotype, but renal gamma-ENaC cleavage was altered. In response to a low-salt diet, particularly with high potassium intake, Tmprss2-/- mice increased plasma aldosterone significantly more than wild-type mice to achieve a similar reduction of renal sodium excretion. Importantly, the stimulatory effect of trypsin on renal tubular ENaC currents was much more pronounced in Tmprss2-/- mice than that in wild-type mice. This indicated the presence of incompletely cleaved and less active channels at the cell surface of TMPRSS2-deficient tubular epithelial cells.ConclusionsTMPRSS2 contributes to proteolytic ENaC activation in mouse kidney in vivo.

Regulation of ENaC in the distal nephron is essential for sodium homeostasis and blood pressure control. A unique feature of ENaC is its complex proteolytic processing leading to channel activation due to release of inhibitory tracts from its α- and γ-subunits. In particular, fully cleaved γ-ENaC is critical for high channel activity. However, the physiologically relevant proteases involved remain elusive. Using Xenopus laevis oocytes and H441 airway epithelial cells, we recently demonstrated that TMPRSS2 proteolytically activates ENaC by cleaving the channel’s γ-subunit ( J Biol Chem 2022 Jun;298(6):102004). Here we investigate whether TMPRSS2 also contributes to proteolytic ENaC regulation in the kidney. Using mRNA sequencing, we demonstrated that mouse cortical collecting duct (mCCD cl1 ) cells express TMPRSS2 at a high level unaffected by aldosterone, an important ENaC stimulating hormone. TMPRSS2 knockdown by CRISPR/Cas9 technology significantly reduced fully cleaved γ-ENaC in membrane-enriched fractions of mCCD cl1 cell lysates (n=5). Apical application of chymotrypsin significantly stimulated ENaC-mediated short circuit current ( I SC ) in TMPRSS2-knockdown cells, but not in control cells (2.0 ± 1.1 μA/cm² vs. 0.2 ± 0.6 μA/cm²; n=13-14; p<0.001; mean ± SD). Moreover, the stimulatory effect of aldosterone on I SC was impaired in TMPRSS2-knockdown cells compared to control cells (9.1 ± 4.0 μA/cm² vs. 18.3 ± 4.0 μA/cm²; n=7-8; p<0.01). After aldosterone treatment I SC stimulation by chymotrypsin was enhanced in TMPRSS2-knockdown cells (5.3 ± 1.9 μA/cm²; n=7) but undetectable in control cells (−0.8 ± 1.6 μA/cm²; n=8). These findings indicate that TMPRSS2 deficiency impairs proteolytic ENaC activation in mCCD cl1 cells. To confirm this in an in vivo model, we used constitutive TMPRSS2 knockout mice ( Tmprss2 -/- ). Using a combination of RNAscope technology and immunofluorescence, we demonstrated high expression of Tmprss2 mRNA in renal tubular cells, including cells with β-ENaC protein staining. Importantly, the ratio of fully to partially cleaved γ-ENaC was significantly reduced in membrane-enriched fractions of whole-kidney lysates from Tmprss2 -/- mice (n=10). Maintained on a standard diet, Tmprss2 -/- mice had no apparent renal phenotype with plasma aldosterone levels similar to those of Tmprss2 +/+ mice (120 ± 43 pg/ml in Tmprss2 -/- vs. 92 ± 17 pg/mL in Tmprss2 +/+ ; n=7-10). Moreover, the ability of Tmprss2 -/- mice to reduce urinary sodium excretion in response to four days of low sodium diet was fully preserved. However, under low sodium diet plasma aldosterone increased to significantly higher levels in Tmprss2 -/- mice compared to controls (752 ± 146 pg/ml in Tmprss2 -/- vs. 268 ± 55 pg/ml in Tmprss2 +/+ ; n=7-11; p<0.01). This is most likely a compensatory response to compromised proteolytic ENaC activation due to TMPRSS2 deficiency. We conclude that TMPRSS2 plays a physiological role in proteolytic ENaC processing and activation in the kidney. This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 509149993, TRR 374 (subproject A4 to A.I. and C.K.) and the Bayerisches Staatsministerium für Wissenschaft und Kunst (Bavarian Ministry of Art and Science), project VI-Corona-Forschung (subproject 07 to M.B. and T.G). A.K. was supported by the Interdisziplinäres Zentrum für klinische Forschung (IZKF) Erlangen. S.W. was supported by the Bundesministerium für Bildung und Forschung (BMBF, Federal Ministry of Education and Research, Germany), project SENSE-CoV2, 01KI20172A. P.S. and F.A. were funded by the IZKF Tübingen. The authors declare no conflicts of interest. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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-ENaC 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 that coexpression of TMPRSS2 stimulated ENaC-mediated whole-cell currents by approximately threefold, likely because of 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 that 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 that TMPRSS2 is likely to contribute to proteolytic ENaC activation in epithelial tissues in vivo.

How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel's α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to in vitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 β (GSK3β). Therefore, we investigated the effect of DYRK2 on ENaC activity in outside-out patches of Xenopus laevis oocytes heterologously expressing rat ENaC. DYRK2 included in the pipette solution significantly increased ENaC activity. In contrast, GSK3β had an inhibitory effect. Replacing S621 in αENaC with alanine (S621A) abolished the effects of both kinases. A S617A mutation reduced the inhibitory effect of GKS3β but did not prevent ENaC activation by DYRK2. Our findings suggest that phosphorylation of S621 activates ENaC and primes S617 for subsequent phosphorylation by GSK3β resulting in channel inhibition. In proof-of-concept experiments, we demonstrated that DYRK2 can also stimulate ENaC currents in microdissected mouse distal nephron, whereas GSK3β inhibits the currents.

Bile acids (BAs) are known to be important regulators of intestinal motility and epithelial fluid and electrolyte transport. Over the past two decades, significant advances in identifying and characterizing the receptors, transporters, and ion channels targeted by BAs have led to exciting new insights into the molecular mechanisms involved in these processes. Our appreciation of BAs, their receptors, and BA-modulated ion channels as potential targets for the development of new approaches to treat intestinal motility and transport disorders is increasing. In the current review, we aim to summarize recent advances in our knowledge of the different BA receptors and BA-modulated ion channels present in the gastrointestinal system. We discuss how they regulate motility and epithelial transport, their roles in pathogenesis, and their therapeutic potential in a range of gastrointestinal diseases.

Regulated Na+ transport in the distal nephron is of fundamental importance to fluid and electrolyte homeostasis. Further upstream, Na+ is the principal driver of secondary active transport of numerous organic and inorganic solutes. In the distal nephron, Na+ continues to play a central role in controlling the body levels and concentrations of a more select group of ions, including K+, Ca++, Mg++, Cl−, and HCO3−, as well as water. Also, of paramount importance are transport mechanisms aimed at controlling the total level of Na+ itself in the body, as well as its concentrations in intracellular and extracellular compartments. Over the last several decades, the transporters involved in moving Na+ in the distal nephron, and directly or indirectly coupling its movement to that of other ions have been identified, and their interrelationships brought into focus. Just as importantly, the signaling systems and their components—kinases, ubiquitin ligases, phosphatases, transcription factors, and others—have also been identified and many of their actions elucidated. This review will touch on selected aspects of ion transport regulation, and its impact on fluid and electrolyte homeostasis. A particular focus will be on emerging evidence for site-specific regulation of the epithelial sodium channel (ENaC) and its role in both Na+ and K+ homeostasis. In this context, the critical regulatory roles of aldosterone, the mineralocorticoid receptor (MR), and the kinases SGK1 and mTORC2 will be highlighted. This includes a discussion of the newly established concept that local K+ concentrations are involved in the reciprocal regulation of Na+-Cl− cotransporter (NCC) and ENaC activity to adjust renal K+ secretion to dietary intake.

ROMK-mediated renal K + secretion is essential for maintaining K + balance and requires a lumen negative transepithelial potential critically dependent on activity of the ENaC. Using microdissected distal mouse tubules, we demonstrated that baseline apical ROMK activity is high in the DCT2/CNT. Aldosterone-independent baseline ENaC activity is also high in the DCT2/CNT and downregulated by a low-K + diet, which highlights the important role of the DCT2/CNT in regulating K + secretion in an aldosterone-independent manner.