ENaC Subunits Research Articles

Enhancing Osmotic Energy Harvesting Through Supramolecular Design of Oxygen‐Functionalized MXene with Biomimetic Ion Channels

AbstractReverse electrodialysis (RED) technology, relying on ion‐selective permeability membranes (ISPM), offers a direct means of harnessing osmotic energy from the salinity difference between seawater and freshwater. Critical technical challenges include limitations in ISPM's immobile charge carriers, transmembrane ionic internal resistance, and durability in water. Drawing inspiration from the subunit structure of the epithelial sodium channel (ENaC), an ISPM assembled using a supramolecular engineering strategy is introduced. This innovative approach enables the synergistic action of multiple molecular building blocks to mimic the distribution of ENaC subunit structures, strategically planning the placement of immobile charge carriers on nanofluidic channels. The design incorporates nano‐confined oxygen‐rich functionalized MXene (O‐MXene) and high‐strength polymer backbone aramid fibers to construct 3D nanofluidic channels with a high surface charge density. Enhanced by a bonding network of sodium carboxymethylcellulose, the ISPM achieved an exceptional output power density of 21.7 W m−2 and 46.0% energy conversion efficiency in a RED half‐cell system using natural seawater and river water, surpassing current technologies. This research not only advances RED technology but also provides biomimetic customization concepts for ISPM design across various applications, including flow batteries, fuel cells, and related fields.

A conserved interaction between the GRIP and finger domains in ENaC subunit is critical for proteolytic activation

The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the lung and the aldosterone-sensitive distal nephron. ENaC is a heterotrimer comprising ⍺βγ or δβγ subunits. Though sharing similar structural folds as other ENaC/Degenerin family members, ENaC subunits have a unique extracellular GRIP (Gating Release of Inhibition by Proteolysis) domain, and the a and g subunits have polybasic sites that can be cleaved by proteases. Aldosterone promotes double cleavage of the α and γ subunits at these sites, leading to release of the first two b-strands of the GRIP domain (P1 and P2) and channel activation. Structural and sequence conservation suggest that the interaction between P1 and the channel are mediated by a Pro-Leu motif in P1 and a conserved Trp residue in helix α2 of the finger domain. We hypothesized that this interaction is critical for the inhibitory properties of the imbedded tract, defined by P1, and thus channel activation upon release. To test our hypothesis, we substituted the conserved Trp with Ala or Phe in each of the human ENaC subunits and performed two-electrode voltage clamp experiments in Xenopus oocytes to measure the effects on function. Compared to wild type channels, we found that ⍺W251F, βW218A, βW218F, γW229F, and δW396F had no effect on amiloride-sensitive currents. Interestingly, δW396A decreased amiloride-sensitive current by 20% (p=0.01). However, amiloride-sensitive currents were greatly enhanced for Ala mutations in subunits subject to cleavage: ⍺W251A (p=0.02) and γW229A (p=0.01). In line with channel activation, ⍺W251A weakened sensitivity to extracellular Na+ (Na+ self-inhibition), and γW229A completely lost its sensitivity to both extracellular Na+ and chymotrypsin. We also tested the effect of inhibitory peptides (α8 and γ11) corresponding to the imbedded inhibitory tracts (i.e., P1 of the GRIP domain) of the ⍺ and γ subunits. ⍺W251A was insensitive to 10 μM ⍺8 compared to wild type ENaC (85% inhibition), and γW229A was insensitive to 100 μM γ11 compared to wild type ENaC (40% inhibition). Collectively, our results provide evidence for the idea that the interaction between the Pro-Leu motif in GRIP domain’s P1 β-strand and the conserved Trp in the finger domain’s α2 helix is critical for the autoinhibitory properties of P1, and therefore activation upon P1 release after proteolysis of the ⍺ and γ subunits. R01 DK125439 Kashlan (PI) ENaC Regulation by Biliary Factors. This is the full abstract presented at the American Physiology Summit 2024 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.

Effect of SARS-CoV-2 S protein on the proteolytic cleavage of the epithelial Na+ channel ENaC.

Severe cases of COVID-19 are characterized by development of acute respiratory distress syndrome (ARDS). Water accumulation in the lungs is thought to occur as consequence of an exaggerated inflammatory response. A possible mechanism could involve decreased activity of the epithelial Na+ channel, ENaC, expressed in type II pneumocytes. Reduced transepithelial Na+ reabsorption could contribute to lung edema due to reduced alveolar fluid clearance. This hypothesis is based on the observation of the presence of a novel furin cleavage site in the S protein of SARS-CoV-2 that is identical to the furin cleavage site present in the alpha subunit of ENaC. Proteolytic processing of αENaC by furin-like proteases is essential for channel activity. Thus, competition between S protein and αENaC for furin-mediated cleavage in SARS-CoV-2-infected cells may negatively affect channel activity. Here we present experimental evidence showing that coexpression of the S protein with ENaC in a cellular model reduces channel activity. In addition, we show that bidirectional competition for cleavage by furin-like proteases occurs between 〈ENaC and S protein. In transgenic mice sensitive to lethal SARS-CoV-2, however, a significant decrease in gamma ENaC expression was not observed by immunostaining of lungs infected as shown by SARS-CoV2 nucleoprotein staining.

Tmprss2 maintains epithelial barrier integrity and transepithelial sodium transport.

The mouse cortical collecting duct cell line presents a tight epithelium with regulated ion and water transport. The epithelial sodium channel (ENaC) is localized in the apical membrane and constitutes the rate-limiting step for sodium entry, thereby enabling transepithelial transport of sodium ions. The membrane-bound serine protease Tmprss2 is co-expressed with the alpha subunit of ENaC. αENaC gene expression followed the Tmprss2 expression, and the absence of Tmprss2 resulted not only in down-regulation of αENaC gene and protein expression but also in abolished transepithelial sodium transport. In addition, RNA-sequencing analyses unveiled drastic down-regulation of the membrane-bound protease CAP3/St14, the epithelial adhesion molecule EpCAM, and the tight junction proteins claudin-7 and claudin-3 as also confirmed by immunohistochemistry. In summary, our data clearly demonstrate a dual role of Tmprss2 in maintaining not only ENaC-mediated transepithelial but also EpCAM/claudin-7-mediated paracellular barrier; the tight epithelium of the mouse renal mCCD cells becomes leaky. Our working model proposes that Tmprss2 acts via CAP3/St14 on EpCAM/claudin-7 tight junction complexes and through regulating transcription of αENaC on ENaC-mediated sodium transport.

Increased ENaC-mediated liquid absorption across vitamin-D deficient human airway epithelia.

Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased SCNN1G and ATP1B1 mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive Isc and Gt without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive Isc and Gt. Furthermore, trypsin nominally increased Isc produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease.NEW & NOTEWORTHY It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.

Proteolytic Activation of the Epithelial Sodium Channel (ENaC): Its Mechanisms and Implications.

Epithelial sodium channel (ENaC) are integral to maintaining salt and water homeostasis in various biological tissues, including the kidney, lung, and colon. They enable the selective reabsorption of sodium ions, which is a process critical for controlling blood pressure, electrolyte balance, and overall fluid volume. ENaC activity is finely controlled through proteolytic activation, a process wherein specific enzymes, or proteases, cleave ENaC subunits, resulting in channel activation and increased sodium reabsorption. This regulatory mechanism plays a pivotal role in adapting sodium transport to different physiological conditions. In this review article, we provide an in-depth exploration of the role of proteolytic activation in regulating ENaC activity. We elucidate the involvement of various proteases, including furin-like convertases, cysteine, and serine proteases, and detail the precise cleavage sites and regulatory mechanisms underlying ENaC activation by these proteases. We also discuss the physiological implications of proteolytic ENaC activation, focusing on its involvement in blood pressure regulation, pulmonary function, and intestinal sodium absorption. Understanding the mechanisms and consequences of ENaC proteolytic activation provides valuable insights into the pathophysiology of various diseases, including hypertension, pulmonary disorders, and various gastrointestinal conditions. Moreover, we discuss the potential therapeutic avenues that emerge from understanding these mechanisms, offering new possibilities for managing diseases associated with ENaC dysfunction. In summary, this review provides a comprehensive discussion of the intricate interplay between proteases and ENaC, emphasizing the significance of proteolytic activation in maintaining sodium and fluid balance in both health and disease.

The B1 H + -ATPase ( Atp6v1b1 ) Subunit in Non-Type A Intercalated Cells is Required for Driving Pendrin Activity and the Renal Defense Against Alkalosis.

In the kidney, the B1 H + -ATPase subunit is mostly expressed in intercalated cells (IC). Its importance in acid-secreting type A ICs is evident in patients with inborn distal renal tubular acidosis and ATP6V1B1 mutations. However, the protein is also highly expressed in alkali-secreting non-type A ICs where its function is incompletely understood. We demonstrate in Atp6v1b1 knock out mice that the B1 subunit is critical for the renal response to defend against alkalosis during an alkali load or chronic furosemide treatment. These findings highlight the importance of non-type A ICs in maintaining acid-base balance in response to metabolic challenges or commonly used diuretics. Non-type A ICs in the collecting duct system express the luminal Cl - /HCO 3- exchanger pendrin and apical and/or basolateral H + -ATPases containing the B1 subunit isoform. Non-type A ICs excrete bicarbonate during metabolic alkalosis. Mutations in the B1 subunit (ATP6V1B1) cause distal renal tubular acidosis due to its role in acid secretory type A ICs. The function of B1 in non-type A ICs has remained elusive. We examined the responses of Atp6v1b1-/- and Atp6v1b1+/+ mice to an alkali load and to chronic treatment with furosemide. An alkali load or 1 week of furosemide resulted in a more pronounced hypokalemic alkalosis in male ATP6v1b1-/- versus Atp6v1b1+/+ mice that could not be compensated by respiration. Total pendrin expression and activity in non-type A ICs of ex vivo microperfused cortical collecting ducts were reduced, and β2 -adrenergic stimulation of pendrin activity was blunted in ATP6v1b1-/- mice. Basolateral H + -ATPase activity was strongly reduced, although the basolateral expression of the B2 isoform was increased. Ligation assays for H + -ATPase subunits indicated impaired assembly of V 0 and V 1 H + -ATPase domains. During chronic furosemide treatment, ATP6v1b1-/- mice also showed polyuria and hyperchloremia versus Atp6v1b1+/+ . The expression of pendrin, the water channel AQP2, and subunits of the epithelial sodium channel ENaC were reduced. Our data demonstrate a critical role of H + -ATPases in non-type A ICs function protecting against alkalosis and reveal a hitherto unrecognized need of basolateral B1 isoform for a proper H + -ATPase complexes assembly and ability to be stimulated.

Kidney-Specific Membrane-Bound Serine Proteases CAP1/Prss8 and CAP3/St14 Affect ENaC Subunit Abundances but Not Its Activity.

The serine proteases CAP1/Prss8 and CAP3/St14 are identified as ENaC channel-activating proteases in vitro, highly suggesting that they are required for proteolytic activation of ENaC in vivo. The present study tested whether CAP3/St14 is relevant for renal proteolytic ENaC activation and affects ENaC-mediated Na+ absorption following Na+ deprivation conditions. CAP3/St14 knockout mice exhibit a significant decrease in CAP1/Prss8 protein expression with altered ENaC subunit and decreased pNCC protein abundances but overall maintain sodium balance. RNAscope-based analyses reveal co-expression of CAP3/St14 and CAP1/Prss8 with alpha ENaC in distal tubules of the cortex from wild-type mice. Double CAP1/Prss8; CAP3/St14-deficiency maintained Na+ and K+ balance on a Na+-deprived diet, restored ENaC subunit protein abundances but showed reduced NCC activity under Na+ deprivation. Overall, our data clearly show that CAP3/St14 is not required for direct proteolytic activation of ENaC but for its protein abundance. Our study reveals a complex regulation of ENaC by these serine proteases on the expression level rather than on its proteolytic activation.

Abstract 098: Collecting Duct-derived (pro)renin Receptor Contributes To 2-kidney, 1-clip-induced Renovascular Hypertension And Ischemic Nephropathy In Mice

The 2-kidney, 1-clip (2K1C) Goldblatt model features overactivation of the systemic renin-angiotensin-aldosterone system (RAAS) due to increased renin release from juxtaglomerular cells. However, no prior study functionally evaluates possible involvement of the intrarenal RAAS in this model. Within the kidney, (pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD) implicated in regulation of the intrarenal RAAS under physio-pathological conditions. In the present study, we employed a mouse model of CD-specific deletion of PRR (CD PRR KO) to examine the role of CD PRR in pathogenesis of 2K1C-induced renovascular hypertension and ischemic nephropathy and to further explore the underlying mechanism. Floxed and CD PRR KO mice were subjected to sham-operation or 2K1C procedure, followed by analysis of blood pressure, renal injury, and indices of the RAAS, as well as the expression of subunits of ENaC. Clipping-induced hypertension and renal injury were both attenuated in CD PRR KO mice as compared with floxed controls (MAP on day24: Floxed/2K1C 148.2±3.9 mm Hg vs. CD PRR KO/2K1C 136.9±2.5 mm Hg, n=6, p < 0.05) (urinary albumin/creatinine: Floxed/2K1C 67.5±6.4 mg/g vs. CD PRR KO/2K1C 30.9±3.5 mg/g, n=6, p < 0.01). Of note, the reductions of clipping-induced protein abundance of fibronectin and α-SMA in CD PRR KO mice were 70% and 55%, respectively. Similarly, clipping induced paralleled increases in renal mRNA expression of IL-1β, TNF-α, MCP-1 and TGF-β1 in floxed mice, which were all blunted in CD PRR KO mice. The protective phenotype of the null mice was paralleled with suppressed intrarenal renin and aldosterone (Aldo) levels. In contrast, clipping-induced enhancement of circulating renin and Aldo remained unchanged between the genotypes. Moreover, renal medullary α-ENaC mRNA and protein expression was elevated by clipping in floxed mice, which was respectively blunted 52% and 54% in CD PRR KO mice. Together, these results suggest that the activation of CD PRR stimulates components of the intrarenal RAAS and renal medullary α-ENaC, which result in increased tubular sodium reabsorption and thus contribute to 2K1C-induced renovascular hypertension and ischemic nephropathy.

Abstract P3019: During Salt-sensitive Hypertension, CD8+ T Cells Increase Renal Sodium Transporters, NKCC2 And ENaC

Hypertension originates in part from impaired salt handling by the kidneys. However, the exact identity of this kidney defect remains unknown. As established recently, the adaptive immunity, T cells in particular, acts as an important contributor to the pathogenesis of hypertension. In this regard, our laboratory has reported that CD8+ T cells (CD8Ts) adoptively transferred from salt-sensitive hypertensive mice to normotensive mice stimulate Sodium Chloride Cotransporter (NCC) increasing sodium retention. However, whether other sodium transporters within the nephron are affected by CD8Ts and contribute to this pathogenesis of hypertension remains uncharacterized. We hypothesize CD8Ts will also have an impact on other sodium channels. Through in vivo studies, we employed a CD8T-adoptive transfer model as described in our previous publications. The kidneys from mice receiving adoptive transfer of hypertensive CD8Ts were harvested and protein levels of NKCC, αENaC, βENaC, and γENaC were analyzed by western blot. In vitro, we co-cultured mouse CD8Ts with Collecting Duct epithelial cells (CDs, M1 cells) to examine the direct interaction between CD8Ts and CDs in media containing various levels of corticosteroids. Similar western blots were performed after this co-culture, as well as measurement of sodium retention through an intracellular sodium indicator and flow cytometry, as reported previously. In vivo, we found that the kidneys of mice receiving adoptive transfer of hypertensive CD8Ts demonstrated higher expression of NKCC, βENaC and γENaC, but no change in αENaC. As for in vitro, we further found that in charcoal-stripped co-culture conditions, pre-activated CD8Ts stimulate higher expression of ENaC subunits and ENaC-mediated sodium retention in M1 cells. However, these effects are masked in culture conditions with physiological or with additional corticosteroid levels. Overall, our results suggest that, as a potential kidney defect, hypertensive CD8Ts strongly stimulate NCC and NKCC in the kidney to mediate excessive salt retention and any effect of CD8Ts in increasing ENaC may be masked in physiologic conditions.

A mild and transient form of autosomal recessive pseudohypoaldosteronism type 1 caused by a novel mutation in the SCNN1A gene.

We investigate the genetic etiology in a cohort of patients with a clinical, biochemical, and hormonal profile suggestive of a mild and transient form of pseudohypoaldosteronism type 1 (PHA1). Twelve patients with PHA1 from four different families with clinical and biochemical data were analyzed. The coding regions of NR3C2 and SCNN1A genes were sequenced. Human α-epithelial sodium channel (ENaC) wild-type (wt), αPhe226Cys and αPhe226Ser ENaC variants were expressed in Xenopus laevis oocytes to evaluate ENaC activity. The protein expression of α-ENaC wt and mutants was determined by Western blot. All patients were homozygotes for the p.Phe226Cys mutation of the α subunit of ENaC. In functional studies in X. laevis oocytes, p.Phe226Cys caused a significant reduction of ENaC activity (83% reduction), reduced the number of active ENαC mutant channels, and reduced the basal open probability compared with wt. Quantitative Western blot analysis revealed that the reduced activity of ENαC mutant channels was due to a reduced ENaC protein expression for the αPhe226Cys compared with wt. We present 12 patients from four different families with a mild and transient autosomal recessive PHA1 due to a novel homozygous missense mutation in the SCNN1A gene. Functional studies showed that the p.Phe226Cys substitution mutation in ENaC leads to a partial loss of function resulting mainly from both a decrease in the intrinsic ENaC activity and a reduction in channel expression at the protein level. The partial loss of ENaC function could explain the mild phenotype, variable expressivity, and the transient course of the disorder in these patients.NEW & NOTEWORTHY This paper demonstrates that mild autosomal recessive pseudohypoaldosteronism type 1 (PHA1) due to p.Phe226Cys missense mutation in the extracellular domain of ENαC α subunit can be transient, with phenotypic variability even with the normal sweat test, and incomplete penetrance. Functional studies explain the phenotype and denote the importance of the location on the extracellular domain of the SCNN1A p.Phe226Cys mutation for the intrinsic ENaC activity and the channel expression at the protein level.

CD8+ T cells stimulate renal sodium transporters contributing to salt-sensitive hypertension

Impaired salt handling by the kidneys acts as one source of hypertension. However, the exact identity of this kidney defect remains unknown. As established recently, the adaptive immunity, T cells in particular, acts as an important contributor to the pathogenesis of hypertension. In this regard, our laboratory has reported that CD8+ T cells (CD8Ts), when adoptively transferred from salt-sensitive hypertensive mice to normotensive mice, stimulate Sodium Chloride Cotransporter (NCC). Through this stimulation, sodium retention occurs, and salt-sensitive hypertension develops. However, whether other sodium transporters within the nephron are affected by hypertensive CD8Ts and contribute to this pathogenesis of hypertension remains uncharacterized. We hypothesize hypertensive CD8Ts will also have an impact on other sodium channels. Here, we demonstrate the impact of hypertensive CD8Ts on sodium-potassium-co-transporter (NKCC) and epithelial sodium channel (ENaCs) in the kidney and the potential of these regulations contributing to salt-sensitive hypertension.Through in vivo studies, we employed a CD8T-adoptive transfer model as described in our previous publications. The kidneys from mice receiving adoptive transfer of hypertensive CD8Ts were harvested and protein levels of NKCC, αENaC, βENaC, and γENaC were analyzed by western blot. With in vitro studies, we co-cultured mouse CD8Ts with Collecting Duct epithelial cells (CDs, M1 cells) to examine the direct interaction between CD8Ts and CDs in media containing various levels of corticosteroids. Similar western blots were performed after this co-culture, as well as measurement of sodium retention through an intracellular sodium indicator and flow cytometry, as reported previously.In the results of in vivo studies, we found that the kidneys of mice receiving adoptive transfer of hypertensive CD8Ts demonstrated higher expression of NKCC, βENaC and γENaC, but no change in αENaC. In the results from in vitro studies, we further found that in charcoal-stripped co-culture conditions, pre-activated CD8Ts stimulate higher expression of ENaC subunits and ENaC-mediated sodium retention in M1 cells. However, these effects are masked in culture conditions with physiological hormone levels or with additional corticosteroids.Overall, our results suggest that, as a potential kidney defect, hypertensive CD8Ts strongly stimulate NCC and NKCC in the kidney to mediate excessive salt retention. Although CD8Ts themselves can increase ENaC, this effect might be masked in physiologic conditions. NIH R01-HL146713 (Mu), AHA 15BGIA25730047 (Mu), and SPaT T32-GM10699 (Deck) 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.

Epithelial sodium channel (ENaC) in GtoPdb v.2023.1

Epithelial sodium channel (ENaC) in GtoPdb v.2023.1

Pendrin abundance, subcellular distribution, and function are unaffected by either αENaC gene ablation or by increasing ENaC channel activity

The intercalated cell Cl−/HCO3− exchanger, pendrin, modulates ENaC subunit abundance and function. Whether ENaC modulates pendrin abundance and function is however unknown. Because αENaC mRNA has been detected in pendrin-positive intercalated cells, we hypothesized that ENaC, or more specifically the αENaC subunit, modulates intercalated cell function. The purpose of this study was therefore to determine if αENaC is expressed at the protein level in pendrin-positive intercalated cells and to determine if αENaC gene ablation or constitutively upregulating ENaC activity changes pendrin abundance, subcellular distribution, and/or function. We observed diffuse, cytoplasmic αENaC label in pendrin-positive intercalated cells from both mice and rats, with much lower label intensity in pendrin-negative, type A intercalated cells. However, while αENaC gene ablation within principal and intercalated cells of the CCD reduced Cl− absorption, it did not change pendrin abundance or subcellular distribution in aldosterone-treated mice. Further experiments used a mouse model of Liddle’s syndrome to explore the effect of increasing ENaC channel activity on pendrin abundance and function. The Liddle’s variant did not increase either total or apical plasma membrane pendrin abundance in aldosterone-treated or in NaCl-restricted mice. Similarly, while the Liddle’s mutation increased total Cl− absorption in CCDs from aldosterone-treated mice, it did not significantly affect the change in Cl− absorption seen with pendrin gene ablation. We conclude that in rats and mice, αENaC localizes to pendrin-positive ICs where its physiological role remains to be determined. While pendrin modulates ENaC abundance, subcellular distribution, and function, ENaC does not have a similar effect on pendrin.

Abstract P2-16-07: Overexpression of the alpha subunit of the epithelial sodium channel in MDAMB231 breast cancer cells reduces cell migration and proliferation

Abstract Breast cancer is the most common cancer diagnosed in women worldwide and leads to 600,000+ deaths annually. The vast majority of breast cancer-related deaths are attributed to metastasis from the primary site in the breast to visceral organs or the brain. Therefore, understanding how these cells achieve metastasis is vital. Ion channels, such as the epithelial sodium channel (ENaC), are emerging as new targets for cancer research due to their role in regulating cell functions such as the process that allows cancer cells to undergo phenotypic changes necessary for metastasis. Ion channel dysregulation may be crucial in cancer development due to the ion channels’ potential role in controlling cancer cell characteristics such as migration and proliferation. There is limited research investigating the role ENaC may have in breast cancer. We have shown that more migratory, mesenchymal breast cancer cell lines have reduced mRNA and protein expression of ENaC compared to more epithelial breast cancer cell lines. Importantly, higher expression levels of the pore-forming alpha-ENaC subunit is correlated with an increased breast cancer survival in patients (n=1705) included in the SCAN-B data set. Bioinformatic analysis, using the SCAN-B data set, showed an increase in the expression of alpha-ENaC mRNA correlated with lower expression levels of migration and proliferation markers, suggesting ENaC has a role in breast cancer cell proliferation and migration. Therefore, this research examines the influence of stably overexpressing the pore-forming subunit of ENaC, alpha-ENaC, in breast cancer cells on their cell migration and proliferation ability. We hypothesised that increasing the expression of ENaC will restore the cells to a more epithelial state, thus reducing cell migration and proliferation. MDAMB231 breast cancer cells were engineered via a stable transfection to overexpress alpha-ENaC or to express an empty vector control. Several clones were isolated and expanded and using RTqPCR, alpha-ENaC mRNA overexpression was confirmed to consistently have 200-fold increased mRNA expression compared to control cells (n=9, p=0.0006). An increase in alpha-ENaC protein expression was also confirmed via western blotting. Using an EdU assay the alpha-ENaC overexpressing cells were shown to have a significant reduction in proliferation compared to the control cell line (n=3, p=0.0048). The alpha-ENaC overexpressing cells showed reduced migratory ability when examined in two established migration assays. In scratch wound assays, the alpha-ENaC overexpressing cells had a significant reduction in cell migration at 24 hours post scratch (n=4, p=0.0001). In Boyden chamber assays, the number of alpha-ENaC-overexpressing cells that migrated through the membrane was significantly reduced compared to the control cell line (n=4, p=0.02). Our results suggest that increased sodium entry into the cell through ENaC has a role in preventing the development of metastatic breast cancer cells, and highlights ENaC as a potential target for future breast cancer therapies. Citation Format: Sarah McQueen, Adam Ware, Heather Cunliffe, Fiona McDonald. Overexpression of the alpha subunit of the epithelial sodium channel in MDAMB231 breast cancer cells reduces cell migration and proliferation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-07.

Proteolytic Activity against the Distal Polybasic Tract of the Gamma Subunit of the Epithelial Sodium Channel ENaC in Nephrotic Urine.

Experimental nephrotic syndrome in mice leads to proteolytic activation of the epithelial sodium channel ENaC, possibly involving the distal polybasic tract of its γ-subunit (183RKRK). We sought to determine if urine samples from both nephrotic mice and a cohort of patients with acute nephrotic syndrome contain a specific proteolytic activity against this region of γ-ENaC. A peptide substrate consisting of amino acids 180-194 of murine γ-ENaC was N-terminally coupled to a fluorophore, yielding AMCA-FTGRKRKISGKIIHK. The substrate was incubated with nephrotic urine samples from mice as well as patients with or without the serine protease inhibitor, aprotinin. The digested peptides were separated on a reverse phase HPLC and detected with a fluorescence detector (350/450 nm). Peptide masses of the peaks were determined with a MALDI-TOF mass spectrometer. In addition, urinary proteolytic activity was quantitated using AMC-coupled substrates reflecting different cleavage sites within the polybasic tract. No significant proteolytic activity against the substrate was found in the urine of healthy humans or mice. Incubation with urine samples of nephrotic patients (n = 8) or mice subjected to three different models of experimental nephrotic syndrome (n = 4 each) led to cleavage of the substrate within the polybasic tract prevented by the serine protease inhibitor aprotinin. The most dominant cleavage product was FTGRKR in both species, which was confirmed using quantitative measurements with FTGRKR- AMC. Nephrotic urine from both humans and mice contains aprotinin-sensitive proteolytic activity against the distal polybasic tract of γ-ENaC, reflecting excretion of active proteases in the urine or proteasuria.

Abstract P247: Altered Renal Sodium Transporters Expression Is Associated With Cholinergic-mediated Salt Sensitive Hypertension In Young Spontaneously Hypertensive Rats

High sodium diet is a major contributing factor and has been associated with inflammation in patients suffering from hypertension. Systemic infusion of nicotine induces renal inflammation and the development of premature hypertension in young spontaneously hypertensive rats (SHR) and leads to enhanced expression of the renal Na-K-Cl cotransporter (NKCC2). Sodium sensitivity also plays a role in the development of cholinergic hypertension. We hypothesized that these findings were related to alterations in renal sodium transporters expression. We continuously infused SHR and Wistar Kyoto (WKY, normotensive control) rats with saline or nicotine (15mg/kg/day) and fed either a low-salt (LSD, 0.01% NaCl) or high-salt (HSD, 4% NaCl) diet for 2 weeks. Renal homogenates were assessed by western blot for the presence of the epithelial sodium channel (ENaC), NKCC2, Na-Cl co-transporter (NCC) and Serum and glucocorticoid regulated kinase 1 (SGK-1). Nicotine infusion in WKY induced 1.9-fold increase (p=0.003) in renal expression of SGK-1 under LSD, compared to saline-infused controls. The sharp increase in SGK-1 in nicotine-infused WKY on LSD was accompanied by a 1.9-fold increase in NCC (p=0.004), a 1.3-fold increase in the cleaved alpha ENaC, a 1.2-fold increase in the uncleaved alpha ENaC, and a 1.5-fold increase in the beta-ENaC (p=0.02). Both saline and nicotine infused WKY showed very little expression of renal NKCC2 in either LSD or HSD. There was no effect of either diet or nicotine infusion on expression of SGK-1, NCC, or ENaC subunits in SHR. Nicotine had no effect on NKCC2 expression in SHR on LSD, but HSD alone induced a 2.25-fold increase in renal NKCC2 expression compared to LSD in saline infused SHR (p=0.006). Nicotine infusion further induced a 58% increase in renal NKCC2 expression for SHR fed HSD (p=0.004). Here we show an effect of nicotine to appropriately increase the expression of NCC and ENaC in WKY under LSD and a trend towards decreased expression under HSD. In contrast, nicotine not only has no effect on NCC and ENaC under LSD in SHR but leads to a paradoxical increase in NKCC2 under HSD. We conclude that inappropriate regulation of renal sodium transporters likely plays a role in cholinergic-mediated renal inflammation and hypertension.

Epithelial Sodium Channel δ Subunit Is Expressed in Human Arteries and Has Potential Association With Hypertension.

Elevated expression and increased activity of vascular epithelial sodium channel (ENaC) can result in vascular dysfunction in small animal models. However, there is limited or no knowledge on expression and function of ENaC channels in human vasculature. Hence, this study explored the expression and function of ENaC in human arteries and their association with hypertension. Human internal mammary artery (IMA) and aorta were obtained from cardiovascular patients undergoing coronary artery bypass graft surgery. Expression of the ENaC subunit was analyzed by polymerase chain reaction, Western blot, and immunohistochemistry. ENaC function was observed by patch-clamp electrophysiology in endothelial cells isolated from IMA. Levels of ENaC subunit expression levels were compared between arteries from normotensive, uncontrolled hypertensive, and controlled hypertensive patients. For the first time, expression of α, β, γ, and δ was detected at mRNA and protein levels in human IMA and aorta. Single-channel patch-clamp recordings identified both αβγ- and δβγ-like channel conductance in primary endothelial cells isolated and cultured from IMA. Reduced expression of the δ subunit was observed in controlled hypertensive IMA, whereas reduced expression of γ-ENaC was observed in controlled hypertensive aorta. These data suggest that functional ENaC channels are expressed in human arteries and their expression levels are associated with hypertension.

Relationship between ENaC Regulators and SARS-CoV-2 Virus Receptor (ACE2) Expression in Cultured Adult Human Fungiform (HBO) Taste Cells.

In addition to the α, β, and γ subunits of ENaC, human salt-sensing taste receptor cells (TRCs) also express the δ-subunit. At present, it is not clear if the expression and function of the ENaC δ-subunit in human salt-sensing TRCs is also modulated by the ENaC regulatory hormones and intracellular signaling effectors known to modulate salt responses in rodent TRCs. Here, we used molecular techniques to demonstrate that the G-protein-coupled estrogen receptor (GPER1), the transient receptor potential cation channel subfamily V member 1 (TRPV1), and components of the renin-angiotensin-aldosterone system (RAAS) are expressed in δ-ENaC-positive cultured adult human fungiform (HBO) taste cells. Our results suggest that RAAS components function in a complex with ENaC and TRPV1 to modulate salt sensing and thus salt intake in humans. Early, but often prolonged, symptoms of COVID-19 infection are the loss of taste, smell, and chemesthesis. The SARS-CoV-2 spike protein contains two subunits, S1 and S2. S1 contains a receptor-binding domain, which is responsible for recognizing and binding to the ACE2 receptor, a component of RAAS. Our results show that the binding of a mutated S1 protein to ACE2 decreases ACE2 expression in HBO cells. We hypothesize that changes in ACE2 receptor expression can alter the balance between the two major RAAS pathways, ACE1/Ang II/AT1R and ACE2/Ang-(1–7)/MASR1, leading to changes in ENaC expression and responses to NaCl in salt-sensing human fungiform taste cells.

Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer.

Lung cancer is one of the most common cancer types in the world. Despite existing treatment strategies, overall patient survival remains low and new targeted therapies are required. Acidification of the tumor microenvironment drives the growth and metastasis of many cancers. Acid sensors such as acid-sensing ion channels (ASICs) may become promising targets for lung cancer therapy. Previously, we showed that inhibition of the ASIC1 channels by a recombinant analogue of mambalgin-2 from Dendroaspis polylepis controls oncogenic processes in leukemia, glioma, and melanoma cells. Here, we studied the effects and molecular targets of mambalgin-2 in lung adenocarcinoma A549 and Lewis cells, lung transformed WI-38 fibroblasts, and lung normal HLF fibroblasts. We found that mambalgin-2 inhibits the growth and migration of A549, metastatic Lewis P29 cells, and WI-38 cells, but not of normal fibroblasts. A549, Lewis, and WI-38 cells expressed different ASIC and ENaC subunits, while normal fibroblasts did not at all. Mambalgin-2 induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma cells. In line, acidification-evoked inward currents were observed only in A549 and WI-38 cells. Gene knockdown showed that the anti-proliferative and anti-migratory activity of mambalgin-2 is dependent on the expression of ASIC1a, α-ENaC, and γ-ENaC. Using affinity extraction and immunoprecipitation, mambalgin-2 targeting of ASIC1a/α-ENaC/γ-ENaC heteromeric channels in A549 cells was shown. Electrophysiology studies in Xenopus oocytes revealed that mambalgin-2 inhibits the ASIC1a/α-ENaC/γ-ENaC channels with higher efficacy than the ASIC1a channels, pointing on the heteromeric channels as a primary target of the toxin in cancer cells. Finally, bioinformatics analysis showed that the increased expression of ASIC1 and γ-ENaC correlates with a worse survival prognosis for patients with lung adenocarcinoma. Thus, the ASIC1a/α-ENaC/γ-ENaC heterotrimer can be considered a marker of cell oncogenicity and its targeting is promising for the design of new selective cancer therapeutics.