Regulation Of Epithelial Na+ Channel Research Articles

Epithelial Na + Channels Function as Extracellular Sensors.

The epithelial Na + channel (ENaC) resides on the apical surfaces of specific epithelia in vertebrates and plays a critical role in extracellular fluid homeostasis. Evidence that ENaC senses the external environment emerged well before the molecular identity of the channel was reported three decades ago. This article discusses progress toward elucidating the mechanisms through which specific external factors regulate ENaC function, highlighting insights gained from structural studies of ENaC and related family members. It also reviews our understanding of the role of ENaC regulation by the extracellular environment in physiology and disease. After familiarizing the reader with the channel's physiological roles and structure, we describe the central role protein allostery plays in ENaC's sensitivity to the external environment. We then discuss each of the extracellular factors that directly regulate the channel: proteases, cations and anions, shear stress, and other regulators specific to particular extracellular compartments. For each regulator, we discuss the initial observations that led to discovery, studies investigating molecular mechanism, and the physiological and pathophysiological implications of regulation. © 2024 American Physiological Society. Compr Physiol 14:5407-5447, 2024.

A mathematical model of ENaC and Slc26a6 regulation by CFTR in salivary gland ducts.

Cystic fibrosis (CF) is a genetic disease caused by the mutations of cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis transmembrane conductance regulator gene. Cftr is a critical ion channel expressed in the apical membrane of mouse salivary gland striated duct cells. Although Cftr is primarily a Cl- channel, its knockout leads to higher salivary Cl- and Na+ concentrations and lower pH. Mouse experiments show that the activation of Cftr upregulates epithelial Na+ channel (ENaC) protein expression level and Slc26a6 (a 1Cl-:2[Formula: see text] exchanger of the solute carrier family) activity. Experimentally, it is difficult to predict how much the coregulation effects of CFTR contribute to the abnormal Na+, Cl-, and [Formula: see text] concentrations and pH in CF saliva. To address this question, we construct a wild-type mouse salivary gland model and simulate CFTR knockout by altering the expression levels of CFTR, ENaC, and Slc26a6. By reproducing the in vivo and ex vivo final saliva measurements from wild-type and CFTR knockout animals, we obtain computational evidence that ENaC and Slc26a6 activities are downregulated in CFTR knockout in salivary glands.NEW & NOTEWORTHY This paper describes a salivary gland mathematical model simulating the ion exchange between saliva and the salivary gland duct epithelium. The novelty lies in the implementation of CFTR regulating ENaC and Slc26a6 in a CFTR knockout gland. By reproducing the experimental saliva measurements in wild-type and CFTR knockout glands, the model shows that CFTR regulates ENaC and Slc26a6 anion exchanger in salivary glands. The method could be used to understand the various cystic fibrosis phenotypes.

Cellular and Molecular Mechanisms Regulating the Normal Physiological Function of ENaC in Salt-Sensitive Hypertension

High blood pressure is a significant risk factor for cardiovascular disease. Many details about the molecular mechanisms that regulate blood pressure are unknown. The Epithelial Na + Channel (ENaC) is the final arbiter of Na + excretion in the kidneys. Appropriate renal sodium excretion is a key factor in the regulation of blood pressure. Abnormalities in ENaC function have been directly linked to several human diseases of blood pressure. The kinase, casein kinase II (CKII), phosphorylates ENaC but the physiological importance of this is obscure. The CKII phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. Phosphorylation of Nav and KCNQ channels by CKII acts as a molecular “switch” favoring the binding of ankyrin-3 (Ank-3). The binding of Ank-3 facilitates the proper membrane localization of these channels increasing their activity. Here, we tested the premise that phosphorylation of ENaC by CKII within “anchor” motifs is necessary and sufficient for Ank-3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion and blood pressure. We tested this hypothesis using endpoint measurements including analyses of ENaC activity and trafficking to the plasma membrane, and quantification of CKII-dependent ENaC-Ank3 binding. This was addressed by combining total internal reflection fluorescence microscopy with fluorescence recovery after photo bleaching (TIRF/FRAP) to follow movement of ENaC toward the plasma membrane in living cells and electrophysiology of ENaC activity in split-open collecting ducts from principal cell-specific CKII and Ank3 knockout mice. We also used whole animal physiological studies of sodium excretion and blood pressure in knockout mice to understand the physiological consequences of CKII and Ank3 regulation of ENaC. In addition, scanning mutagenesis was used to identify key residues in the overlapping CKII and Ank3 sites in ENaC. Furthermore, we tested whether CKII influences Ank-3 binding to β-ENaC subunit using fluorescence resonance energy transfer (FRET). Findings showed that disrupting CKII signaling and abrogation of the CKII phosphorylation and Ank3 binding site within ENaC decreases channel trafficking toward the plasma membrane and activity, promotes improper sodium excretion, and decreases blood pressure in KO mice. These results showed how and when CKII phosphorylation of ENaC functions as a “switch” to favor Ank-3 binding to increase channel activity to include having a detailed understanding of the mechanisms mediating this regulation, and a rich appreciation of the physiological consequences of such regulation. This work was supported by National Institutes of Health (NIH) grants R01 HL139841 and R01 DK113816. 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.

Impact of 5'-AMP-activated protein kinase (AMPK) on Epithelial Sodium Channels (ENaCs) in human sperm

Hyperpolarization is associated with decreased intracellular Na+ concentration through the closure of the epithelial Na+ channels (ENaCs) during capacitation. 5'-AMP-activated protein kinase (AMPK) is involved in the regulation of Na+ transport by reducing ENaC-β abundance in the plasma membrane in somatic cells. However, it is not known whether AMPK acts on ENaCs in sperm. The aim of the present study was to analyze the role of AMPK activation in the regulation of ENaC and to examine its relationship with capacitation-associated hyperpolarization of human sperm. Human sperm were treated with AICAR (AMPK activator) in non-capacitating and capacitating conditions. AMPK activity and ENaC-β concentration were evaluated by ELISA. Flow cytometry was used to measure tyrosine phosphorylation, hyperpolarization, intracellular Na+ concentration and acrosome reaction. Immunofluorescence staining was carried out to analyze the distribution of ENaC-β and CD46 in sperm. We found that induction of capacitation triggered AMPK phosphorylation. AMPK activation by AICAR increased tyrosine phosphorylation. AICAR decreased ENaC-β levels, mainly localized at the principal-piece of the flagellum, resulting in lower intracellular Na+ concentration and increased hyperpolarization of the plasma membrane. Altogether, these data provide evidence that AMPK activation is involved in capacitation-associated hyperpolarization by reducing ENaC abundance in human sperm.

Cellular and Molecular Mechanisms Regulating the Normal Physiological Function of the Epithelial Sodium Channel

High blood pressure is a significant risk factor for cardiovascular disease. Many details about the molecular mechanisms that regulate blood pressure are unknown. The Epithelial Na+ Channel (ENaC) is the final arbiter of Na+ excretion in the kidneys (see attached Figure). Appropriate renal sodium excretion is a key factor in the regulation of blood pressure. Abnormalities in ENaC function have been directly linked to several human diseases of blood pressure. The kinase, casein kinase II (CKII), phosphorylates ENaC but the physiological importance of this is obscure. The CKII phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. Phosphorylation of Nav and KCNQ channels by CKII acts as a molecular “switch” favoring the binding of ankyrin‐3 (Ank‐3). The binding of Ank‐3 facilitates the proper membrane localization of these channels increasing their activity. Here, we tested the premise that phosphorylation of ENaC by CKII within “anchor” motifs is necessary and sufficient for Ank‐3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion and blood pressure (see attached Figure). We tested this hypothesis using endpoint measurements including analyses of ENaC activity and trafficking to the plasma membrane, and quantification of CKII‐dependent ENaC‐Ank3 binding. This was addressed by combining total internal reflection fluorescence microscopy with fluorescence recovery after photo bleaching (TIRF/FRAP) to follow movement of ENaC toward the plasma membrane in living cells and electrophysiology of ENaC activity in split‐open collecting ducts from principal cell‐specific CKII and Ank3 knockout mice. We also used whole animal physiological studies of sodium excretion and blood pressure in knockout mice to understand the physiological consequences of CKII and Ank3 regulation of ENaC. In addition, scanning mutagenesis was used to identify key residues in the overlapping CKII and Ank3 sites in ENaC. Furthermore, we tested whether CKII influences Ank‐3 binding to β‐ENaC subunit using fluorescence resonance energy transfer (FRET). Findings showed that disrupting CKII signaling and abrogation of the CKII phosphorylation and Ank3 binding site within ENaC decreases channel trafficking toward the plasma membrane and activity, promotes improper sodium excretion, and decreases blood pressure in KO mice. These results showed how and when CKII phosphorylation of ENaC functions as a “switch” to favor Ank‐3 binding to increase channel activity to include having a detailed understanding of the mechanisms mediating this regulation, and a rich appreciation of the physiological consequences of such regulation.

Functional assessment of PIP 2 regulation of ENaC using an optogenetic dimerization assay

The epithelial Na+ channel (ENaC) plays a key role in Na+ transport in epithelial linings to include the lung, colon and kidney. In the distal kidney tubules, ENaC regulates Na+ reabsorption and blood volume. Thus, dysfunctions in signaling pathways regulating ENaC activity are linked to hypertension or hypotension. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a target of the G protein coupled receptor P2Y2 pathway, and is necessary for the proper function of ENaC. This nonvoltage-gated trimeric channel is comprised of α, β, and γ subunits. We recently described two intracellular PIP2 binding sites on the N termini of β-, and γ-ENaC, with moderate μM affinity. Here, we report the functional effects on ENaC containing a combination of mutations to those PIP2 binding sites, by controlled depletion of PIP2. We used a CIBN/CRY2-5-ptase optogenetic dimerization system to deplete PIP2 levels in HEK293 cells transiently expressing wild type (wt) ENaC or the mutant ENaC constructs. CoroNa Green, a fluorescent Na+ indicator, was used to monitor ENaC activity by tracking the relative intracellular Na+ levels. Upon optogenetic-controlled depletion of PIP2, Na+ levels decreased in cells expressing wt ENaC. Mutations to the PIP2 sites of ENaC were expected to have no change in Na+ levels upon PIP2 depletion due to the disruption of PIP2 binding. As a control, mutations to non-PIP2 binding sites were included, and were expected to have decreased Na+ levels similar to wt ENaC. Interestingly, mutation of each independent PIP2 site resulted in only a small decrease of intracellular Na+, compared to wt ENaC. However, mutations throughout the entire N-terminus of β-ENaC, including the PIP2 binding site, resulted in a significant increase of Na+ upon PIP2 depletion. These data suggest that the residues surrounding the PIP2 binding sites play a significant role in the affinity of PIP2 for ENaC. The role of these other domains in PIP2 binding is still under investigation.

Gs‐DREADD Regulation of ENaC

Activity of the epithelial Na+ channel (ENaC) fine-tunes renal Na+ excretion and consequently, contributes to the normal control of blood pressure. ENaC is expressed in the apical membrane of principal cells. Importantly, ENaC is activated by the hormone vasopressin via the V2 receptor, which is coupled to the heterotrimeric Gs protein. Stimulation of the V2 receptor rapidly activates adenylyl cyclase to increase intracellular cAMP levels. We have recently developed a mouse model with targeted expression of the Gs designer receptors exclusively activated by designer drugs (DREADD) in renal principal cells (GsD). Moreover, this mouse line has a luciferase reporter downstream of a cAMP response element binding protein site. As such, it allows in vivo reporting of cAMP levels. DREADD receptors are activated by clozapine N-oxide (CNO), which normally is an inert drug with no biological activity in the absence of DREADD receptors. We test here if CNO affects the activity of ENaC to influence renal salt and water excretion in GsD mice similarly to vasopressin. In these mice, we longitudinally monitored CNO-stimulated cAMP levels in principal cells using an IVIS Lumina XR. In parallel, we quantified CNO-dependent ENaC activity and renal sodium excretion. We observed that CNO increased ENaC activity in principal cells in freshly isolated tubules from GsD mice compared to control mice. Metabolic cages experiments demonstrated that CNO treatment (0.1 mg/kg) increased cAMP levels concomitantly with reducing sodium excretion similarly to dDAVP (1 µg) in GsD mice. Finally, in COS-7 cells expressing murine ENaC tagged with YFP, CPT-cAMP increased translocation of ENaC to the plasma membrane. Here we demonstrated the utility of a new principal cell-specific Gs-DREADD mouse line. This new mouse model will be an important tool for investigating the effects of abnormal cAMP signaling in principal cells possibly informing understanding of the cellular mechanism underpinning conditions like polycystic kidney disease.

Abstract P229: Site-1 Protease-derived Soluble (pro) Renin Receptor Mediates Aldosterone-induced Enac Activation In The Collecting Duct

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na + channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), generated by site-1 protease (S1P), a newly identified PRR cleavage protease, in ENaC regulation, and further tested its relevance to Aldo signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM for 24 h induced a significant increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique ( I eq : 7.5 ± 0.7 μA/cm 2 in sPRR group vs. 3.5 ± 0.5 μA/cm 2 in vehicle group, n = 6, p < 0.01) . In primary cultured rat IMCD cells, the same sPRR-His treatment induced a 1.7 fold increase in protein expression of the α-subunit but not β- or γ-subunit of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The upregulation of α-ENaC transcription depended on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Using chamber determination of short-circuit current showed that Aldo-induced ENaC activity was almost completely abolished by PF-429242 (PF), a S1P inhibitor, and the response was restored by supplement of sPRR-His ( I eq : 7.2 ± 0.7 μA/cm 2 in Aldo group vs. 5.0 ± 0.3 μA/cm 2 in Aldo/PF group vs. 6.8 ± 0.3 μA/cm 2 in Aldo/PF/sPRR-His group, n = 5, p < 0.05). Medium sPRR was elevated by Aldo and inhibited by PF. Male C57BL/6 mice were pretreated with PF (30 mg/kg/day) or vehicle via minipump, followed by 3 days of aldosterone (0.2 mg/kg/day via a second minipump). Amiloride-sensitive Na+ current in freshly isolated CCD as measured by using patch clamp lower in Aldo + PF group than in Aldo group. Together, these results support an essential role of S1P-derived sPRR in mediating Aldo-induced ENaC activation.

NOX4-dependent regulation of ENaC in hypertension and diabetic kidney disease.

NADPH oxidase 4 (NOX4) is the most abundant NOX isoform in the kidney; however, its importance for renal function has only recently emerged. The NOX4-dependent pathway regulates many factors essential for proper sodium handling in the distal nephron. However, the functional significance of this pathway in the control of sodium reabsorption during the initiation of chronic kidney disease is not established. The goal of this study was to test Nox4-dependent ENaC regulation in two models: SS hypertension and STZ-induced type 1 diabetes. First, we showed that genetic ablation of Nox4 in Dahl salt-sensitive (SS) rat attenuated a high-salt (HS)-induced increase in epithelial Na+ channel (ENaC) activity in the cortical collecting duct. We also found that H2 O2 upregulated ENaC activity, and H2 O2 production was reduced in both the renal cortex and medulla in SSNox4-/- rats fed an HS diet. Second, in the streptozotocin model of hyperglycemia-induced renal injury ENaC activity in hyperglycemic animals was elevated in SS but not SSNox4-/- rats. NaCl cotransporter (NCC) expression was increased compared to healthy controls, while expression values between SS and SSNox4-/- groups were similar. These data emphasize a critical contribution of the NOX4-mediated pathway in maladaptive upregulation of ENaC-mediated sodium reabsorption in the distal nephron in the conditions of HS- and hyperglycemia-induced kidney injury.

Upregulation of Renal ENaC Activity in Spontaneously Hypertensive Rats Is Independent of Aldosterone

Spontaneously hypertensive rat (SHR) has been widely used as a model to interrogate the contribution of the kidney to the pathogenesis of essential hypertension in humans. Elevation of blood pressure in SHR has been associated with altered renal function, since predisposition to hypertension can be transferred with a transplanted kidney into a normotensive Wistar‐Kyoto (WKY) strain. Renal defects result in excessive water and sodium retention leading to increased circulating volume and blood pressure. Abnormally elevated Na+ reabsorption in SHR is associated with the overactive renin‐angiotensin‐aldosterone system (RAAS) and with the increased abundance of the renal epithelial Na+ channel (ENaC) responsible for final regulation of sodium reuptake in the kidney and pivotal for long‐term blood pressure control. The existing data on regulation of ENaC by RAAS components in SHR is inconsistent and molecular determinants of excessive ENaC activation remain largely obscure. In the present study, we tested the efficacy of aldosterone antagonism to inhibit ENaC activity, decrease renal Na+ reabsorption and reduce blood pressure in SHR of both sexes. Where necessary, age‐matched (12–15 week‐old) WKY rats were used as controls. Consistent with previously published data, enzyme immunoassay analysis revealed elevated circulating aldosterone levels and semi‐quantitative immunoblotting – higher expression of renal ENaC protein in SHR when compared to WKY controls. Patch‐clamp experiments in split‐opened collecting ducts showed that ENaC activity was moderately increased in SHR when compared to WKY. Systemic administration of aldosterone antagonist, spironolactone (30 mg/kgBW·day) did not affect renal ENaC activity or protein abundance in SHR. Aldosterone antagonism did not alter the expression of sodium‐chloride co‐transporter (NCC) in the kidneys of SHR. Tail‐cuff plethysmography showed that treatment with spironolactone at doses 30–200 mg/kgBW·day for 2 weeks failed to decrease blood pressure in SHR and did not result in any detectable changes in urinary sodium excretion. We did not observe any sex‐specific differences pertaining to ENaC activity or responsiveness to spironolactone in SHR males and females. Thus, we conclude that aldosterone is not a primary driver of excessive ENaC‐dependent renal Na+ reabsorption in SHR.

Paraoxonase 3 functions as a chaperone to decrease functional expression of the epithelial sodium channel

The paraoxonase (PON) family comprises three highly conserved members: PON1, PON2, and PON3. They are orthologs of Caenorhabditis elegans MEC-6, an endoplasmic reticulum-resident chaperone that has a critical role in proper assembly and surface expression of the touch-sensing degenerin channel in nematodes. We have shown recently that MEC-6 and PON2 negatively regulate functional expression of the epithelial Na+ channel (ENaC), suggesting that the chaperone function is conserved within this family. We hypothesized that other PON family members also modulate ion channel expression. Pon3 is specifically expressed in the aldosterone-sensitive distal tubules in the mouse kidney. We found here that knocking down endogenous Pon3 in mouse cortical collecting duct cells enhanced Na+ transport, which was associated with increased γENaC abundance. We further examined Pon3 regulation of ENaC in two heterologous expression systems, Fisher rat thyroid cells and Xenopus oocytes. Pon3 coimmunoprecipitated with each of the three ENaC subunits in Fisher rat thyroid cells. As a result of this interaction, the whole-cell and surface abundance of ENaC α and γ subunits was reduced by Pon3. When expressed in oocytes, Pon3 inhibited ENaC-mediated amiloride-sensitive Na+ currents, in part by reducing the surface expression of ENaC. In contrast, Pon3 did not alter the response of ENaC to chymotrypsin-mediated proteolytic activation or [2-(trimethylammonium)ethyl]methanethiosulfonate-induced activation of αβS518Cγ, suggesting that Pon3 does not affect channel open probability. Together, our results suggest that PON3 regulates ENaC expression by inhibiting its biogenesis and/or trafficking.

Soluble (pro)renin receptor regulation of ENaC involved in aldosterone signaling in cultured collecting duct cells.

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na+ channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), the cleavage product of PRR in ENaC regulation, and further tested its relevance to aldosterone signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM within minutes induced a significant and transient increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique. The acute ENaC activation was blocked by the NADPH oxidase 1/4 inhibitor GKT137892 and siRNA against Nox4 but not the β-catenin inhibitor ICG-001. In primary rat inner medullary collecting duct cells, administration of sPRR-His at 10 nM for 24 h induced protein expression of the α-subunit but not β- or γ-subunits of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The transcriptional activation of α-ENaC was dependent on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Ussing chamber determination of short-circuit current showed that aldosterone-induced transepithelial Na+ transport was inhibited by the PRR decoy inhibitor PRO20 and PF-429242, an inhibitor of sPRR-generating enzyme site-1 protease, and the response was restored by the addition of sPRR-His. Medium sPRR was elevated by aldosterone and inhibited by PF-429242. Taken together, these results demonstrate that sPRR induces two phases of ENaC activation via distinct mechanisms and functions as a mediator of the natriferic action of aldosterone.

The epithelial Na+ channel α- and γ-subunits are cleaved at predicted furin-cleavage sites, glycosylated and membrane associated in human kidney.

The epithelial Na+ channel (ENaC) is essential for Na+/K+ homeostasis and blood pressure control. Its activity is regulated by proteases in rodents. To gain more information on proteolytic ENaC regulation in humans, we tested the hypotheses that (1) human kidney α- and γ-ENaC subunits are furin-cleaved, glycosylated, and altered by medication that change plasma aldosterone; (2) prostasin-cleaved γ-ENaC is increased in proteinuria, and (3) cleaved ENaC moieties prevail at the membranes and in urinary extracellular vesicles (uEVs). We developed three monoclonal antibodies (mAbs) targeting (1) the neo-epitope generated after furin cleavage in γ-ENaC (mAb-furin); (2) the intact prostasin cleavage-site in γ-ENaC (mAb-intactRKRK), and (3) the α-ENaC subunit (mAb-alpha). Nephrectomy tissue and uEVs were used for immunoblotting and -histochemistry. In human kidney tissue, mAb-furin detected a ≈ 65-70 kDa protein, compatible with furin-cleaved γ-ENaC; mAb-intactRKRK detected full-length (≈ 90-100 kDa) and furin-cleaved (≈ 70-75 kDa) γ-ENaC. mAb-alpha detected a ≈ 50 kDa protein compatible with furin-cleaved α-subunit. Furin-cleaved γ-ENaC was detected predominantly within membrane fractions and deglycosylation shifted full-length γ-ENaC migration ~ 20 kDa. While γ-ENaC uEV levels were below the detection limit, α-ENaC migrated as intact (≈ 75 kDa) and furin-cleaved (≈ 50 kDa) in uEVs. Kidney levels of α- and γ-ENaC in diuretic- (n = 3) and ACE-inhibitor-treated (n = 4) patients were not different from controls (n = 4). Proteinuric patients (n = 6) displayed similar level of furin-cleaved γ-ENaC as controls (n = 4). Cleaved α-ENaC abundance was significantly lower in the kidneys from proteinuria patients. In conclusion, the study demonstrates ENaC cleavage as an event in human kidney that could contribute to physiological regulation and pathophysiological activation of ENaC.

AMPK phosphorylation of the β1Pix exchange factor regulates the assembly and function of an ENaC inhibitory complex in kidney epithelial cells.

The metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC), a key regulator of salt reabsorption by the kidney and thus total body volume and blood pressure. Recent studies have suggested that AMPK promotes the association of p21-activated kinase-interacting exchange factor-β1 β1Pix, 14-3-3 proteins, and the ubiquitin ligase neural precursor cell expressed developmentally downregulated protein (Nedd)4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and ENaC degradation. Functional β1Pix is required for ENaC inhibition by AMPK and promotes Nedd4-2 phosphorylation and stability in mouse kidney cortical collecting duct cells. Here, we report that AMPK directly phosphorylates β1Pix in vitro. Among several AMPK phosphorylation sites on β1Pix detected by mass spectrometry, Ser71 was validated as functionally significant. Compared with wild-type β1Pix, overexpression of a phosphorylation-deficient β1Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both β1Pix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a β1Pix-Δ602-611 deletion tract mutant unable to bind 14-3-3 proteins decreased the interaction between Nedd4-2 and 14-3-3 proteins, suggesting that 14-3-3 binding to β1Pix is critical for the formation of a β1Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both β1Pix and Nedd4-2 to 14-3-3 proteins was reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of β1Pix at Ser71, which promotes 14-3-3 interactions with β1Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.

MST3 is involved in ENaC-mediated hypertension.

Liddle syndrome is an inherited form of human hypertension caused by increasing epithelial Na+ channel (ENaC) expression. Increased Na+ retention through ENaC with subsequent volume expansion causes hypertension. In addition to ENaC, the Na+-K+-Cl- cotransporter (NKCC) and Na+-Cl- symporter (NCC) are responsible for Na+ reabsorption in the kidneys. Several Na+ transporters are evolutionarily regulated by the Ste20 kinase family. Ste20-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 phosphorylate downstream NKCC2 and NCC to maintain Na+ and blood pressure (BP) homeostasis. Mammalian Ste20 kinase 3 (MST3) is another member of the Ste20 family. We previously reported that reduced MST3 levels were found in the kidneys in spontaneously hypertensive rats and that MST3 was involved in Na+ regulation. To determine whether MST3 is involved in BP stability through Na+ regulation, we generated a MST3 hypomorphic mutation and designated MST3+/- and MST3-/- mice to examine BP and serum Na+ and K+ concentrations. MST3-/- mice exhibited hypernatremia, hypokalemia, and hypertension. The increased ENaC in the kidney played roles in hypernatremia. The reabsorption of more Na+ promoted more K+ secretion in the kidney and caused hypokalemia. The hypernatremia and hypokalemia in MST3-/- mice were significantly reversed by the ENaC inhibitor amiloride, indicating that MST3-/- mice reabsorbed more Na+ through ENaC. Furthermore, Madin-Darby canine kidney cells stably expressing kinase-dead MST3 displayed elevated ENaC currents. Both the in vivo and in vitro results indicated that MST3 maintained Na+ homeostasis through ENaC regulation. We are the first to report that MST3 maintains BP stability through ENaC regulation.

High mobility group box‐1 increases epithelial sodium channel activity and inflammation in small airway epithelial cells

In Cystic Fibrosis (CF) patients, inherited defects in anion transport leads to hyperactive epithelial Na+ channel (ENaC) activity. ENaC dysfunction in CF lung is known to cause airway surface liquid dehydration. Thick and dry mucus, in turn, leads to an inflammatory lung phenotype that often occurs without detection of infection in CF patients. Many investigative groups have reported that airway tissue and fluid from murine models exhibiting the CF lung phenotype (Scnn1b‐Tr mice), as well as CF patients, have increased levels of high mobility group box‐1 (HMGB‐1). HMGB‐1 is a cytokine mediator of inflammation and is a biomarker for severity of CF lung disorder. Herein, we hypothesize that HMGB‐1 plays an important role in the pathogenic regulation of ENaC, inflammation, and fibrosis in sterile airways. To begin testing this hypothesis, we measured transepithelial sodium current (Isc) and single channel ENaC open probability (Po) in human small airway epithelial cells (SAEC) in the presence or absence of 1 mg/mL human HMGB‐1 peptide. Electrophysiological measurements show that HMGB‐1 significantly increased amiloride‐sensitive ENaC Isc from −9.7±1.4 μA/cm2 to −18.4±2.9 μA/cm2 in confluent monolayers of SAEC (n=12; p=0.01) within 30 min. Subsequent amiloride treatment of the same SEAC monolayers decreased Isc 71%. Single channel measurements were conducted in primary SAEC obtained from 4 separate donor lungs in the cell‐attached configuration; HMGB‐1 increased ENaC Po from 0.18±0.02 to 0.35±0.04 (n=17; p<0.01) in single channel analysis. Mean absolute total cell counts were significantly higher in bronchoalveolar lavage fluid (BALF) from mice intraperitoneally (IP) injected with HMBG‐1 (10 mg in 1 mL saline/kg) vs vehicle injected mice. Total immune cell counts were 27.4×103± 8.8×103 (in HMGB‐1) vs. 5.2×103±1.3X103 (in vehicle) injected mice (n=8 counts from 2 independent studies; p<0.05). Masson's Trichrome labeling of IP injected mice show that HMGB‐1 significantly increases pulmonary fibrosis (see Fig 1). Flow cytometric analysis of HMGB‐1 and vehicle injected mice showed significant increase in IL‐1β, IL‐10, IL‐6, IL‐27, IL‐17A, IFN‐β, and GM‐CSF (n=3; p<0.05; see Fig 2). Together, our data suggests that HMGB‐1 plays an important role in the CF lung phenotype; additional studies are underway to strengthen these observations. Impact: Successful completion of our work can lead to novel therapies needed to counteract ENaC dysfunction and inflammation in the CF lung.Support or Funding InformationThis work was supported by NIH 1 R01 HL137033‐01A1 awarded to MH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

β1Pix exchange factor stabilizes the ubiquitin ligase Nedd4-2 and plays a critical role in ENaC regulation by AMPK in kidney epithelial cells

Our previous work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) by promoting its binding to neural precursor cell-expressed, developmentally down-regulated 4-2, E3 ubiquitin protein ligase (Nedd4-2). Here, using MS analysis and in vitro phosphorylation, we show that AMPK phosphorylates Nedd4-2 at the Ser-444 (Xenopus Nedd4-2) site critical for Nedd4-2 stability. We further demonstrate that the Pak-interacting exchange factor β1Pix is required for AMPK-mediated inhibition of ENaC-dependent currents in both CHO and murine kidney cortical collecting duct (CCD) cells. Short hairpin RNA-mediated knockdown of β1Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β1Pix dimerization-deficient mutant unable to bind 14-3-3 proteins (Δ602-611) increased ENaC currents in CCD cells, whereas overexpression of WT β1Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of β1Pix to 14-3-3 proteins in CCD cells. However, the association between Nedd4-2 and 14-3-3 proteins was not consistently affected by AMPK activation, β1Pix knockdown, or overexpression of WT β1Pix or the β1Pix-Δ602-611 mutant. Moreover, we found that β1Pix is important for phosphorylation of the aforementioned Nedd4-2 site critical for its stability. Overall, these findings elucidate novel molecular mechanisms by which AMPK regulates ENaC. Specifically, they indicate that AMPK promotes the assembly of β1Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation.

β1Pix Stabilizes Nedd4‐2 and Plays a Critical Role in ENaC Regulation by AMPK in Kidney Epithelial Cells

The epithelial Na+ channel (ENaC) is expressed in many salt‐reabsorbing epithelia at the apical membrane, including in the aldosterone‐sensitive distal nephron of the kidney where it is the final regulator of Na+ balance, blood volume, and blood pressure. Our previous work has established that the metabolic sensor AMP‐activated protein kinase (AMPK) inhibits ENaC by promoting the binding of the ubiquitin ligase Nedd4‐2 to ENaC. Here, we have further demonstrated that functional β1Pix, a Rho‐GEF signaling protein, is required for AMPK‐dependent ENaC inhibition in CHO cells. Epithelial volt‐ohmmeter measurements were performed to examine the role of β1Pix in regulating ENaC‐dependent currents in polarized kidney cortical collecting duct (CCD) cells. Lentiviral shRNA‐mediated knockdown of β1Pix expression in mpkCCDcl1 cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β1Pix dimerization‐deficient mutant unable to bind 14‐3‐3 proteins (Δ602‐611) increased ENaC currents in mpkCCDc14 cells, whereas overexpression of wild‐type β1Pix had an opposite effect. Through additional immunoblotting and co‐immunoprecipitation studies, we explored the role of β1Pix in the regulation of ENaC by AMPK and the interplay of β1Pix, 14‐3‐3 proteins and Nedd4‐2. Treatment with AMPK activators promoted the binding of β1Pix to 14‐3‐3 proteins in CCD cells. However, the association between Nedd4‐2 and 14‐3‐3 proteins was not significantly altered by AMPK activation, β1Pix knockdown, or overexpression of wild‐type or β1Pix‐Δ602–611 mutant in cells. Moreover, β1Pix knockdown or overexpression of β1Pix‐Δ602–611 inhibited Nedd4‐2 protein expression in CCD cells. This inhibition was associated with a parallel reduction in Nedd4‐2 phosphorylation at Ser‐328, a site that is phosphorylated by several kinases including AMPK which is critical for cellular Nedd4‐2 protein stability. Decreased Nedd4‐2 protein stability in the setting of β1Pix knockdown was confirmed by cycloheximide chase studies. Together, these results suggest that functional β1Pix is important for Nedd4‐2 Ser‐328 phosphorylation and thus Nedd4‐2 stability. Overall, our findings suggest that AMPK activation promotes, potentially via AMPK‐dependent β1Pix phosphorylation, the association of β1Pix, 14‐3‐3 proteins, and Nedd4‐2 into a complex that inhibits ENaC by enhancing Nedd4‐2 binding to ENaC followed by ENaC ubiquitination and targeting for degradation. This regulation of ENaC and other epithelial transport proteins by AMPK may have a pro‐survival role by inhibiting energy‐consuming ion transport processes during conditions of ischemia and other cellular stresses that occur with acute kidney injury.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SPLUNC1 is an allosteric modulator of the epithelial sodium channel.

Cystic fibrosis (CF) is a common genetic disease with significantly increased mortality. CF airways exhibit ion transport abnormalities, including hyperactivity of the epithelial Na+ channel (ENaC). Short-palate lung and nasal epithelial clone 1 (SPLUNC1) is a multifunctional innate defense protein that is secreted into the airway lumen. We have previously demonstrated that SPLUNC1 binds to and inhibits ENaC to maintain fluid homeostasis in airway epithelia and that this process fails in CF airways. Despite this, how SPLUNC1 actually regulates ENaC is unknown. Here, we found that SPLUNC1 caused αγ-ENaC to internalize, whereas SPLUNC1 and β-ENaC remained at the plasma membrane. Additional studies revealed that SPLUNC1 increased neural precursor cell-expressed developmentally down-regulated protein 4-2-dependent ubiquitination of α- but not β- or γ-ENaC. We also labeled intracellular ENaC termini with green fluorescent protein and mCherry, and found that extracellular SPLUNC1 altered intracellular ENaC Forster resonance energy transfer. Taken together, our data indicate that SPLUNC1 is an allosteric regulator of ENaC that dissociates αβγ-ENaC to generate a new SPLUNC1-β-ENaC complex. These data indicate a novel mode for regulating ENaC at the plasma membrane.-Kim, C. S., Ahmad, S., Wu, T., Walton, W. G., Redinbo, M. R., Tarran, R. SPLUNC1 is an allosteric modulator of the epithelial sodium channel.

Regulation of the epithelial Na+ channel by paraoxonase-2

Paraoxonase-2 (PON-2) is a membrane-bound lactonase with unique anti-oxidative and anti-atherosclerotic properties. PON-2 shares key structural elements with MEC-6, an endoplasmic reticulum-resident molecular chaperone in Caenorhabditis elegans MEC-6 modulates the expression of a mechanotransductive ion channel comprising MEC-4 and MEC-10 in touch-receptor neurons. Because pon-2 mRNA resides in multiple rat nephron segments, including the aldosterone-sensitive distal nephron where the epithelial Na+ channel (ENaC) is expressed, we hypothesized that PON-2 would similarly regulate ENaC expression. We observed PON-2 expression in aquaporin 2-positive principal cells of the distal nephron of adult human kidney. PON-2 also co-immunoprecipitated with ENaC when co-expressed in HEK293 cells. When PON-2 was co-expressed with ENaC in Xenopus oocytes, ENaC activity was reduced, reflecting a reduction in ENaC surface expression. MEC-6 also reduced ENaC activity when co-expressed in Xenopus oocytes. The PON-2 inhibitory effect was ENaC-specific, as PON-2 had no effect on functional expression of the renal outer medullary potassium channel. PON-2 did not alter the response of ENaC to extracellular Na+, mechanical shear stress, or α-chymotrypsin-mediated proteolysis, suggesting that PON-2 did not alter the regulation of ENaC by these factors. Together, our data suggest that PON-2 regulates ENaC activity by modulating its intracellular trafficking and surface expression.