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Aberrant anion secretion across the bronchial epithelium is associated with airway disease, most notably in cystic fibrosis. Although the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as the primary source of airway anion secretion, alternative anion transport mechanisms play a contributing role. An alternative anion transporter of growing interest is SLC26A9, a constitutively active chloride channel that has been shown to interact with CFTR and may also contribute to bicarbonate secretion. Interest in SLC26A9 has been fueled by genome-wide association studies that suggest it is a significant modifier of CF disease severity. Despite this growing evidence that SLC26A9 plays an important role in the airway, its presence and function in bronchial epithelia remain poorly understood, in part, because its activity is difficult to separate from the activity of CFTR. Here, we present results using primary human bronchial epithelia (HBE) from multiple patient sources to confirm that SLC26A9 mRNA is present in HBE and that its constitutive channel activity is unaffected by knockdown of CFTR. Furthermore, SLC26A9 and CFTR show differential responses to common inhibitors of anion secretion. Finally, we assess the impact of bicarbonate on the activity of SLC26A9 and CFTR. These results confirm that SLC26A9 is the primary source of constitutive anion secretion across HBE, and should inform future studies focused on activation of SLC26A9 as an alternative anion channel in CF. These results should provide a strong foundation to investigate how single-nucleotide polymorphisms in SLC26A9 modulate airway disease.

Guanylate cyclase 2C (GC‐C), encoded by the GUCY2C gene, is implicated in hereditary early onset chronic diarrhea. Several families with chronic diarrhea symptoms have been identified with autosomal dominant, gain‐of‐function mutations in GUCY2C. We have identified a Mennonite patient with a novel GUCY2C variant (c.2381A > T; p.Asp794Val) with chronic diarrhea and an extensive maternal family history of chronic diarrhea and bowel dilatation. Functional studies including co‐segregation analysis showed that all family members who were heterozygous for this variant had GI‐related symptoms. HEK‐293 T cells expressing the Asp794Val GC‐C variant showed increased cGMP production when stimulated with Escherichia coli heat‐stable enterotoxin STp (HST), which was reversed when 5‐(3‐Bromophenyl)‐5,11‐dihydro‐1,3‐dimethyl‐1H‐indeno[2′,1′:5,6]pyrido[2,3‐d]pyrimidine‐2,4,6(3H)‐trione (BPIPP; a GC‐C inhibitor) was used. In addition, cystic fibrosis transmembrane conductance regulator (CFTR) activity measured with SPQ fluorescence assay was increased in these cells after treatment with HST, indicating a crucial role for CFTR activity in the pathogenesis of this disorder. These results support pathogenicity of the GC‐C Asp794Val variant as a cause of chronic diarrhea in this family. Furthermore, this work identifies potential candidate drug, GC‐C inhibitor BPIPP, to treat diarrhea caused by this syndrome.

SLC26A9, a member of the solute carrier protein family, transports chloride ions across various epithelia. SLC26A9 also associates with other ion channels and transporters linked to human health, and in some cases these heterotypic interactions are essential to support the biogenesis of both proteins. Therefore, understanding how this complex membrane protein is initially folded might provide new therapeutic strategies to overcome deficits in the function of SLC26A9 partners, one of which is associated with Cystic Fibrosis. To this end, we developed a novel yeast expression system for SLC26A9. This facile system has been used extensively with other ion channels and transporters to screen for factors that oversee protein folding checkpoints. As commonly observed for other channels and transporters, we first noted that a substantial fraction of SLC26A9 is targeted for endoplasmic reticulum associated degradation (ERAD), which destroys folding-compromised proteins in the early secretory pathway. We next discovered that ERAD selection requires the Hsp70 chaperone, which can play a vital role in ERAD substrate selection. We then created SLC26A9 mutants and found that the transmembrane-rich domain of SLC26A9 was quite stable, whereas the soluble cytosolic STAS domain was responsible for Hsp70-dependent ERAD. To support data obtained in the yeast model, we were able to recapitulate Hsp70-facilitated ERAD of the STAS domain in human tissue culture cells. These results indicate that a critical barrier to nascent membrane protein folding can reside within a specific soluble domain, one that is monitored by components associated with the ERAD machinery.

TMEM16A, a Ca2+ -activated Cl- channel, contributes to tumor growth in breast cancer and head and neck squamous cell carcinoma (HNSCC). Here, we investigated whether TMEM16A influences the response to EGFR/HER family-targeting biological therapies. Inhibition of TMEM16A Cl- channel activity in breast cancer cells with HER2 amplification induced a loss of viability. Cells resistant to trastuzumab, a monoclonal antibody targeting HER2, showed an increase in TMEM16A expression and heightened sensitivity to Cl- channel inhibition. Treatment of HNSCC cells with cetuximab, a monoclonal antibody targeting EGFR, and simultaneous TMEM16A suppression led to a pronounced loss of viability. Biochemical analyses of cells subjected to TMEM16A inhibitors or expressing chloride-deficient forms of TMEM16A provide further evidence that TMEM16A channel function may play a role in regulating EGFR/HER2 signaling. These data demonstrate that TMEM16A regulates EGFR and HER2 in growth and survival pathways. Furthermore, in the absence of TMEM16A cotargeting, tumor cells may acquire resistance to EGFR/HER inhibitors. Finally, targeting TMEM16A improves response to biological therapies targeting EGFR/HER family members.

Cystic fibrosis (CF) disease is caused by mutations affecting the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in the mucosal side of epithelial tissue. In the airway, dysfunctional CFTR results in a transepithelial osmotic imbalance leading to hyperabsorption of airway surface liquid mucostasis, chronic inflammation, and eventual respiratory failure. Human nasal epithelial cell cultures from healthy and CF donors were used to perform studies of liquid and solute transport dynamics at an air/liquid interface in order to emulate the in vivo airway. Then, these results were used to inform a quantitative systems pharmacology model of airway epithelium describing electrically and chemically driven transcellular ionic transport, contributions of both convective and diffusive paracellular solute transport, and osmotically driven transepithelial water dynamics. Model predictions showed CF cultures, relative to non-CF ones, have increased apical and basolateral water permeabilities, and increase paracellular permeability and transepithelial chemical driving force for a radiolabeled tracer used to track small molecule absorption. These results provide a computational platform to better understand and probe the mechanisms behind the liquid hyperabsorption and small molecule retention profiles observed in the CF airway.

A pathway for CFTR degradation is initiated by Hsp27 which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogs form poly-chains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, PIAS4, which increased WT and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR three-fold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation 3-fold and stabilized mature WT CFTR at the PM. PIAS4 knockdown reduced WT and F508del CFTR expression by 40-50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1in vivoand reduced its conjugation to SUMO-2/3. These SUMO paralog specific effects of PIAS4 were reproducedin vitrousing purified F508del NBD1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ~50%, and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogs determine the fates of WT and mutant CFTRs, and they suggest that a paralog switch during biogenesis can direct these proteins to different outcomes: biogenesis vs. degradation.

cAMP/PKA stimulation elicited posttranslational increases in CFTR expression and the interaction of specific 14-3-3 proteins with phosphorylated sites within the R region. This improved the efficiency of nascent CFTR biogenesis and reduced its interaction with the COPI retrograde retrieval mechanism, making more CFTR available for anion secretion., Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)–regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.

The focus of this review is the regulated trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) in distal compartments of the protein secretory pathway and the question of how changes in CFTR cellular distribution may impact on the functions of polarized epithelial cells. We summarize data concerning the cellular localization and activity of CFTR and attempt to synthesize often conflicting results from functional studies of regulated endocytosis and exocytosis in CFTR-expressing cells. In some instances, findings that are inconsistent with regulated CFTR trafficking may result from the use of overexpression systems or nonphysiological experimental conditions. Nevertheless, judging from data on other transporters, an appropriate cellular context is necessary to support regulated CFTR trafficking, even in epithelial cells. The discovery that disease mutations can influence CFTR trafficking in distal secretory and recycling compartments provides support for the concept that regulated CFTR recycling contributes to normal epithelial function, including the control of apical CFTR channel density and epithelial protein secretion. Finally, we propose molecular mechanisms for regulated CFTR endocytosis and exocytosis that are based on CFTR interactions with other proteins, particularly those whose primary function is membrane trafficking. These models provide testable hypotheses that may lead to elucidation of CFTR trafficking mechanisms and permit their experimental manipulation in polarized epithelial cells.

Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR ), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.

Human bronchial epithelial (HBE) cells exhibit constitutive anion secretion that is absent in cells from cystic fibrosis (CF) patients. The identity of this conductance is unknown, but SLC26A9, a member of the SLC26 family of CF transmembrane conductance regulator (CFTR)-interacting transporters, is found in the human airway and exhibits chloride channel behavior. We sought differences in the properties of SLC26A9 and CFTR expressed in HEK 293 (HEK) cells as a fingerprint to identify HBE apical anion conductances. HEK cells expressing SLC26A9 displayed a constitutive chloride current that was inhibited by the CFTR blocker GlyH-101 (71 +/- 4%, 50 microM) and exhibited a near-linear current-voltage (I-V) relation during block, while GlyH-101-inhibited wild-type (wt)CFTR exhibited a strong inward-rectified (IR) I-V relation. We tested polarized HBE cells endogenously expressing either wt or DeltaF508-CFTR for similar activity. After electrical isolation of the apical membrane using basolateral alpha-toxin permeabilization, wtCFTR monolayers displayed constitutive chloride currents that were inhibited by GlyH-101 (68 +/- 6%) while maintaining a near-linear I-V relation. In the absence of blocker, the addition of forskolin stimulated a current increase having a linear I-V; GlyH-101 blocked 69 +/- 7% of the current and shifted the I-V relation IR, consistent with CFTR activation. HEK cells coexpressing SLC26A9 and wtCFTR displayed similar properties, as well as forskolin-stimulated currents that exceeded the sum of those in cells separately expressing SLC26A9 or wtCFTR, and an I-V relation during GlyH-101 inhibition that was moderately IR, indicating that SLC26A9 contributed to the stimulated current. HBE cells from CF patients expressed SLC26A9 mRNA, but no constitutive chloride currents. HEK cells coexpressing SLC26A9 with DeltaF508-CFTR also failed to exhibit SLC26A9 current. We conclude that SLC26A9 functions as an anion conductance in the apical membranes of HBE cells, it contributes to transepithelial chloride currents under basal and cAMP/protein kinase A-stimulated conditions, and its activity in HBE cells requires functional CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

The innate immune functions of human airways include mucociliary clearance and antimicrobial peptide activity. Both functions may be affected by changes in epithelial ion transport. Interleukin-17A (IL-17A), which has a receptor at the basolateral membrane of airway epithelia, is a T cell cytokine that has been shown to increase mucus secretion and antimicrobial peptide production by human bronchial epithelial (HBE) cells. Furthermore, IL-17A levels are increased in sputum from patients during pulmonary exacerbations of cystic fibrosis. Therefore, we investigated the effects of IL-17A on basal, amiloride-sensitive, and forskolin-stimulated ion transport in mature, well-differentiated HBE cells. Exposure of HBE monolayers to IL-17A for 48 h induced a novel forskolin-stimulated bicarbonate secretion in addition to forskolin-stimulated chloride secretion and resulted in alkalinization of liquid on the mucosal surface of polarized cells. IL-17A-induced bicarbonate secretion was cystic fibrosis transmembrane conductance regulator (CFTR)-dependent, mucosal chloride-dependent, partially Na+-dependent, and sensitive to serosal, but not mucosal, stilbene inhibition. These data suggest that IL-17A modulates epithelial bicarbonate secretion and implicate a mechanism by which airway surface liquid pH changes may be abnormal in cystic fibrosis.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). The most common mutation, ΔF508, omits the phenylalanine residue at position 508 in the first nucleotide binding domain (NBD1) of CFTR. The mutant protein is retained in the endoplasmic reticulum and degraded by the ubiquitin-proteasome system. We demonstrate that expression of NBD1 plus the regulatory domain (RD) of ΔF508 CFTR (ΔFRD) restores the biogenesis of mature ΔF508 CFTR protein. In addition, ΔFRD elicited a cAMP-stimulated anion conductance response in primary human bronchial epithelial (HBE) cells isolated from homozygous ΔF508 CF patients. A protein transduction domain (PTD) could efficiently transduce (∼90%) airway epithelial cells. When fused to a PTD, direct addition of the ΔFRD peptide conferred a dose-dependent, cAMP-stimulated anion efflux to ΔF508 HBE cells. Hsp70 and Hsp90 associated equally with WT and ΔF508 CFTR, whereas nearly twice as much of the Hsp90 cochaperone, Aha1, associated with ΔF508 CFTR. Expression of ΔFRD produced a dose-dependent removal of Aha1 from ΔF508 CFTR that correlated with its functional rescue. These findings indicate that disruption of the excessive association of the cochaperone, Aha1, with ΔF508 CFTR is associated with the correction of its maturation, trafficking and regulated anion channel activity in human airway epithelial cells. Thus, PTD-mediated ΔFRD fragment delivery may provide a therapy for CF.—Sun, F., Mi, Z., Condliffe, S. B., Bertrand, C. A., Gong, X., Lu, X., Zhang, R., Latoche, J. D., Pilewski, J. M., Robbins, P. D., Frizzell, R. A. Chaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epithelia.

Cystic fibrosis is a common lethal genetic disease among Caucasians. The cystic fibrosis gene encodes a cyclic adenosine monophosphate-activated chloride channel (cystic fibrosis transmembrane conductance regulator (CFTR)) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. Mutations in CFTR fall into two broad categories; those that affect protein biosynthesis/stability and traffic to the cell surface and those that cause altered channel kinetics in proteins that reach the cell surface. Here we report a novel mechanism by which mutations in CFTR give rise to disease. N287Y, a mutation within an intracellular loop of CFTR, increases channel endocytosis from the cell surface without affecting either biosynthesis or channel gating. The sole consequence of this novel mutation is to generate a novel tyrosine-based endocytic sequence within an intracellular loop in CFTR leading to increased removal from the cell surface and a reduction in the steady-state level of CFTR at the cell surface.