Kathryn W. Peters

Airway Surface Liquid Volume Regulates ENaC by Altering the Serine Protease-Protease Inhibitor Balance A MECHANISM FOR SODIUM HYPERABSORPTION IN CYSTIC FIBROSIS

Efficient clearance of mucus and inhaled pathogens from the lung is dependent on an optimal airway surface liquid (ASL) volume, which is maintained by the regulated transport of sodium and chloride across the airway epithelium. Accumulating evidence suggests that impaired mucus clearance in cystic fibrosis (CF) airways is a result of ASL depletion caused by excessive Na+ absorption through the epithelial sodium channel (ENaC). However, the cellular mechanisms that result in increased ENaC activity in CF airways are not completely understood. Recently, proteases were shown to modulate the activity of ENaC, but the relevance of this mechanism to the physiologic regulation of ASL volume is unknown. Using primary human airway epithelial cells, we demonstrate that: (i) protease inhibitors are present in the ASL and prevent the activation of near-silent ENaC, (ii) when the ASL volume is increased, endogenous protease inhibitors become diluted, allowing for proteolytic activation of near-silent channels, and (iii) in CF, the normally present near-silent pool of ENaC is constitutively active and the α subunit undergoes increased proteolytic processing. These findings indicate that the ASL volume modulates the activity of ENaC by modification of the serine protease-protease inhibitor balance and that alterations in this balance contribute to excessive Na+ absorption in cystic fibrosis.

Clathrin-mediated Endocytosis of the Epithelial Sodium Channel ROLE OF EPSIN

Here we present evidence that the epithelial sodium channel (ENaC), a heteromeric membrane protein whose surface expression is regulated by ubiquitination, is present in clathrin-coated vesicles in epithelial cells that natively express ENaC. The channel subunits are ubiquitinated and co-immunoprecipitate with both epsin and clathrin adaptor proteins, and epsin, as expected, co-immunoprecipitates with clathrin adaptor proteins. The functional significance of these interactions was evaluated in a Xenopus oocyte expression system where co-expression of epsin and ENaC resulted in a down-regulation of ENaC activity; conversely, co-expression of epsin sub-domains acted as dominant-negative effectors and stimulated ENaC activity. These results identify epsin as an accessory protein linking ENaC to the clathrin-based endocytic machinery thereby regulating the activity of this ion channel at the cell surface.

From CFTR biology toward combinatorial pharmacotherapy: Expanded classification of cystic fibrosis mutations

More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.

Regulation of the amiloride-sensitive epithelial sodium channel by syntaxin 1A.

The first step in transepithelial sodium absorption lies at the apical membrane where the amiloride-sensitive, epithelial sodium channel, ENaC, facilitates sodium entry into the cell. Here we report that the vesicle traffic regulatory (SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)) protein, syntaxin 1A (S1A), inhibits ENaC mediated sodium entry. This inhibitory effect is selective for S1A and is not reproduced by syntaxin 3. The inhibition does not require the membrane anchoring domain of syntaxin 1A. It was reversed by the S1A-binding protein, Munc-18, but not by a Munc-18 mutant, which lacks syntaxin affinity. Immunostaining of epitope-tagged ENaC subunits showed that syntaxin 1A decreases ENaC current by reducing the number of ENaC channels in the plasma membrane; S1A does not interfere with ENaC protein expression. Immunoprecipitation of syntaxin 1A from the sodium-transporting epithelial cell line, A6, co-precipitates ENaC. These findings indicate that syntaxin 1A and other members of the SNARE machinery are involved in the control of plasma membrane ENaC content, and they suggest that SNARE proteins participate in the regulation of sodium absorption in relation to agonist mediated vesicle insertion-retrieval processes.

14-3-3 Isoforms Are Induced by Aldosterone and Participate in Its Regulation of Epithelial Sodium Channels

Aldosterone increases sodium absorption across renal collecting duct cells primarily by increasing the apical membrane expression of ENaC, the sodium entry channel. Nedd4-2, a ubiquitin-protein isopeptide ligase, tags ENaC with ubiquitin for internalization and degradation, but when it is phosphorylated by the aldosterone-induced kinase, SGK1, Nedd4-2 is inhibited and apical ENaC density and sodium absorption increase. We evaluated the hypothesis that 14-3-3 proteins participate in the aldosterone-mediated regulation of ENaC by associating with phosphorylated Nedd4-2. Mouse cortical collecting duct (mCCD) epithelia cultured on filters expressed several 14-3-3 isoforms; this study focused on an isoform whose expression was induced 3-fold by aldosterone, 14-3-3β. In polarized mCCD epithelia, aldosterone elicited significant, time-dependent increases in the expression of α-ENaC, SGK1, phospho-Nedd4-2, and 14-3-3β without altering total Nedd4-2. Aldosterone decreased the interaction of α-ENaC with Nedd4-2, and with similar kinetics increased the association of 14-3-3β with phospho-Nedd4-2. Short interfering RNA-induced knockdown of 14-3-3β blunted the aldosterone-induced increase in α-ENaC expression, returned α-ENaC-Nedd4-2 binding toward prealdosterone levels, and blocked the aldosterone-stimulated increase in transepithelial sodium transport. Incubation of cell extracts with a selective phospho-Nedd4-2 antibody blocked the aldosterone-induced association of 14-3-3β with Nedd4-2, implicating SGK1 phosphorylation at Ser-328 as the primary site of 14-3-3β binding. Our studies show that aldosterone increases the expression of 14-3-3β, which interacts with phospho-Nedd4-2 to block its interaction with ENaC, thus enhancing sodium absorption by increasing apical membrane ENaC density.

Syntaxin 1A inhibits regulated CFTR trafficking in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial cell Cl channel, whose gating activity and membrane trafficking are controlled by cAMP/protein kinase A (PKA)-mediated phosphorylation. CFTR Cl currents are regulated also by syntaxin 1A (A. P. Naren, D. J. Nelson, W. W. Xie, B. Jovov, J. Pevsner, M. K. Bennett, D. J. Benos, M. W. Quick, and K. L. Kirk. Nature 390: 302-305, 1997), a protein best known for its role in membrane trafficking and neurosecretion. To examine the mechanism of syntaxin 1A inhibition, we expressed these proteins in Xenopusoocytes and monitored agonist-induced changes in plasma membrane capacitance and cell surface fluorescence of CFTR that contains an external epitope tag. cAMP stimulation elicited large increases in membrane capacitance and in cell surface labeling of flag-tagged CFTR. Coexpression of CFTR with syntaxin 1A, but not syntaxin 3, inhibited cAMP-induced increases in membrane capacitance and plasma membrane CFTR content. Injection of botulinum toxin/C1 rapidly reversed syntaxins effects on current and capacitance, indicating that they cannot be explained by an effect on CFTR synthesis. Functional expression of other integral membrane proteins, including Na-coupled glucose transporter hSGLT1, inwardly rectified K channel hIK1, P2Y2 nucleotide receptor, and viral hemagglutinin protein, was not affected by syntaxin 1A coexpression. These findings indicate that acute regulation of the number of CFTR Cl channels in plasma membrane is one mechanism by which cAMP/PKA regulates Cl currents. Inhibition of plasma membrane CFTR content by syntaxin 1A is consistent with the concept that syntaxin and other components of the SNARE machinery are involved in regulated trafficking of CFTR.

An Obligatory Heterodimer of 14-3-3β and 14-3-3ε Is Required for Aldosterone Regulation of the Epithelial Sodium Channel

Increased distal nephron sodium absorption in response to aldosterone involves Nedd4-2 phosphorylation, which blocks its ability to ubiquitylate ENaC and increases apical membrane channel density by reducing its endocytosis. Our prior work (Liang, X., Peters, K. W., Butterworth, M. B., and Frizzell, R. A. (2006) J. Biol. Chem. 281, 16323–16332) showed that aldosterone selectively increased 14-3-3 protein isoform expression and that the association of 14-3-3β with phospho-Nedd4-2 was required for sodium transport stimulation. The knockdown of 14-3-3β alone nearly eliminated the response to aldosterone, despite the expression of other 14-3-3 isoforms in cortical collecting duct (CCD) cells. To further examine this marked effect of 14-3-3β knockdown, we evaluated the hypothesis that phospho-Nedd4-2 binding prefers a heterodimer composed of two different 14-3-3 isoforms. We tested this concept in polarized CCD cells using RNA interference and assays of sodium transport and of the interaction of Nedd4-2 with 14-3-3ϵ, a second aldosterone-induced isoform. As observed previously for 14-3-3β knockdown, small interfering RNA-induced reduction of 14-3-3ϵ markedly attenuated aldosterone-stimulated ENaC expression and sodium transport and increased the interaction of Nedd4-2 with ENaC toward prealdosterone levels. After aldosterone induction, 14-3-3β and 14-3-3ϵ were quantitatively co-immunoprecipitated from CCD cell lysates, and the association of both isoforms with Nedd4-2 increased. Finally, the knockdown of either 14-3-3β or 14-3-3ϵ reduced the association of Nedd4-2 with the other isoform. We conclude that the two aldosterone-induced 14-3-3 isoforms, β and ϵ, interact with phospho-Nedd4-2 as an obligatory heterodimer, blocking its interaction with ENaC and thereby increasing apical ENaC density and sodium transport.

Cysteine String Protein Interacts with and Modulates the Maturation of the Cystic Fibrosis Transmembrane Conductance Regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel whose phosphorylation regulates both channel gating and its trafficking at the plasma membrane. Cysteine string proteins (Csps) are J-domain-containing, membrane-associated proteins that have been functionally implicated in regulated exocytosis. Therefore, we evaluated the possibility that Csp is involved in regulated CFTR trafficking. We found Csp expressed in mammalian epithelial cell lines, several of which express CFTR. In Calu-3 airway cells, immunofluorescence colocalized Csp with calnexin in the endoplasmic reticulum and with CFTR at the apical membrane domain. CFTR coprecipitated with Csp from Calu-3 cell lysates. Csp associated with both core-glycosylated immature and fully glycosylated mature CFTRs (bands B and C); however, in relation to the endogenous levels of the B and C bands expressed in Calu-3 cells, the Csp interaction with band B predominated. In vitro protein binding assays detected physical interactions of both mammalian Csp isoforms with the CFTR R-domain and the N terminus, having submicromolar affinities. InXenopus oocytes expressing CFTR, Csp overexpression decreased the chloride current and membrane capacitance increases evoked by cAMP stimulation and decreased the levels of CFTR protein detected by immunoblot. In mammalian cells, the steady-state expression of CFTR band C was eliminated, and pulse-chase studies showed that Csp coexpression blocked the conversion of immature to mature CFTR and stabilized band B. These results demonstrate a primary role for Csp in CFTR protein maturation. The physical interaction of this Hsc70-binding protein with immature CFTR, its localization in the endoplasmic reticulum, and the decrease in production of mature CFTR observed during Csp overexpression reflect a role for Csp in CFTR biogenesis. The documented role of Csp in regulated exocytosis, its interaction with mature CFTR, and its coexpression with CFTR at the apical membrane domain of epithelial cells may reflect also a role for Csp in regulated CFTR trafficking at the plasma membrane.

Role of snare proteins in CFTR and ENaC trafficking

Abstract. The apical membrane ion channels, CFTR and ENaC, undergo regulated trafficking as a means of controlling their plasma membrane density. This provides a mechanism for regulating the Cl and Na conductance properties of epithelial apical membranes, and thus the transepithelial ion transport rates. Physical and functional interactions between these channels and SNARE proteins, in particular syntaxin 1A (S1A), provide a mechanism for linking the known vesicle fusion machinery with this process. In this paper we summarize evidence indicating that the interaction of S1A with CFTR and ENaC reduces channel currents in a syntaxin-isoform-specific manner. The acute cAMP-regulated CFTR trafficking event, which is reported by an increase in membrane capacitance in response to cAMP, is also inhibited by exogenous S1A expression. We tagged both channels with flag epitopes on their extracellular surfaces to monitor their plasma membrane expression as a function of S1A co-expression. The data indicate that the reduction in current caused by S1A is associated with a marked decrease in the amount of CFTR or ENaC detected at the cell surface. These findings suggest that S1A inhibits ion channel insertion into the plasma membrane, either by disrupting the stoichiometry of SNARE protein associations that mediate channel trafficking, or by physically associating with the channels to prevent their insertion. These data link the SNARE machinery to the regulation of apical membrane ion channel density, and suggest that phosphorylation-dependent interactions of these channels with SNARE proteins may acutely regulate this process.

The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct (MPKCCD14) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-β-cyclodextrin added apically to MPKCCD14 cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 ± 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of MPKCCD14 cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.