The function of the cystic fibrosis transmembrane conductance regulator (CFTR) as a Cl؊ channel in the apical membrane of epithelial cells is extensively documented. However, less is known about the molecular determinants of CFTR residence in the apical membrane, basal regulation of its Cl ؊ channel activity, and its reported effects on the function of other transporters. These aspects of CFTR function likely require specific interactions between CFTR and unknown proteins in the apical compartment of epithelial cells. Here we report that CFTR interacts with the recently discovered protein, EBP50 (ERM-binding phosphoprotein 50). EBP50 is concentrated at the apical membrane in human airway epithelial cells, in vivo, and CFTR and EBP50 associate in in vitro binding assays. The CFTR-EBP50 interaction requires the COOH-terminal DTRL sequence of CFTR and utilizes either PDZ1 or PDZ2 of EBP50, although binding to PDZ1 is of greater affinity. Through formation of a complex, the interaction between CFTR and EBP50 may influence the stability and/or regulation of CFTR Cl ؊ channel function in the cell membrane and provides a potential mechanism through which CFTR can affect the activity of other apical membrane proteins.Cystic fibrosis (CF) 1 is a lethal autosomal recessive disease characterized by defects in epithelial ion transport (1). CF is caused by mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a cAMP-regulated Cl Ϫ channel at the apical cell surface (1-3). The CF phenotype includes changes in cellular processes distinct from those involving Cl Ϫ transport, including sodium hyperabsorption and abnormalities in the processing of mucins (4 -6). The most common cause of CF are mutations that lead to the formation of an abnormally folded CFTR protein that does not reach the cell surface (2). Even wild type CFTR is inefficiently transported to the cell surface, with up to 70% of the newly synthesized proteins failing to achieve a stable conformation that escapes quality control mechanisms in the endoplasmic reticulum (2,7,8). Knowledge of the protein-protein interactions that are involved in CFTR-mediated regulation of other epithelial transport proteins, and the interactions that control the trafficking, localization, and regulation of CFTR, is incomplete. Recently, the amino terminus of CFTR was shown to interact with syntaxin 1, with implications both for insertion of CFTR into the plasma membrane and regulation of channel activity (9). Other interactions that stabilize CFTR or regulate its function remain to be identified.Compartmentalization of CFTR in a multiprotein complex might regulate CFTR activity by stabilizing the protein at the cell surface or by increasing the efficiency by which kinases and phosphatases control the channel. The presence of such a complex may also explain how CFTR modulates the activity of other epithelial cell transport proteins. A common mechanism to establish multiprotein complexes is via protein-protein interactions w...

Rationale: Chronic obstructive pulmonary disease is a leading cause of death worldwide, but its pathogenesis is not well understood. Previous studies have shown that airway surface dehydration in b-epithelial Na 1 channel (bENaC)-overexpressing mice caused a chronic lung disease with high neonatal pulmonary mortality and chronic bronchitis in adult survivors. Objectives: The aim of this study was to identify the initiating lesions and investigate the natural progression of lung disease caused by airway surface dehydration. Methods: Lung morphology, gene expression, bronchoalveolar lavage, and lung mechanics were studied at different ages in bENaCoverexpressing mice. Measurements and Main Results: Mucus obstruction in bENaCoverexpressing mice originated in the trachea in the first days of life and was associated with hypoxia, airway epithelial necrosis, and death. In surviving bENaC-overexpressing mice, mucus obstruction extended into the lungs and was accompanied by goblet cell metaplasia, increased mucin expression, and airway inflammation with transient perinatal increases in tumor necrosis factor-a and macrophages, IL-13 and eosinophils, and persistent increases in keratinocytederived cytokine (KC), neutrophils, and chitinases in the lung. bENaCoverexpressing mice also developed emphysema with increased lung volumes, distal airspace enlargement, and increased lung compliance.Conclusions: Our studies demonstrate that airway surface dehydration is sufficient to initiate persistent neutrophilic airway inflammation with chronic airways mucus obstruction and to cause transient eosinophilic airway inflammation and emphysema. These results suggest that deficient airway surface hydration may play a critical role in the pathogenesis of chronic obstructive pulmonary diseases of different etiologies and serve as a target for novel therapies.

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin (ϳ7 m) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nM. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of ϳ250 fmol/min/cm 2 , reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached ϳ1 M independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y 2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.ATP regulates the airway epithelial mucociliary clearance activities that are critical for pulmonary host defense against bacteria which deposit on airway surfaces. ATP activates the G q /phospholipase C-coupled P2Y 2 receptors (P2Y 2 -R), 2 which in turn promotes Cl Ϫ secretion via calcium-activated Cl Ϫ channels (CaCC) (1), inhibits Na ϩ absorption mediated by epithelial sodium channels (2), increases ciliary beat frequency (3), and triggers mucin release (4, 5). Released ATP is rapidly hydrolyzed to ADP, AMP, and adenosine (ADO) by cell-surface nucleotidases. ADO activates G s /adenylyl cyclase-coupled A 2b receptor to promote cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator (CFTR) activation and Cl Ϫ secretion (6, 7). Functional and biochemical evidence indicates that release and subsequent metabolism of ATP on the airway surface contribute to P2Y 2 and A 2b receptor-regulated electrolyte transport and airway surface liquid (ASL) volume homeostasis (8,9).Whereas the roles of ATP and ADO in modulating mucociliary clearance and associated airway epithelial activities have been intensively investigated, it is largely unknown how ATP concentrations in the thin (ϳ7 m) periciliary liquid lining air...

Previous studies in native tissues have produced conflicting data on the localization and metabolic fate of WT and ⌬F508 cystic fibrosis transmembrane regulator (CFTR) in the lung. Combining immunocytochemical and biochemical studies utilizing new high-affinity CFTR mAbs with ion transport assays, we examined both 1) the cell type and region specific expression of CFTR in normal airways and 2) the metabolic fate of ⌬F508 CFTR and associated ERM proteins in the cystic fibrosis lung. Studies of lungs from a large number of normal subjects revealed that WT CFTR protein localized to the apical membrane of ciliated cells within the superficial epithelium and gland ducts. In contrast, other cell types in the superficial, gland acinar, and alveolar epithelia expressed little WT CFTR protein. No ⌬F508 CFTR mature protein or function could be detected in airway specimens freshly excised from a large number of ⌬F508 homozygous subjects, despite an intact ERM complex. In sum, our data demonstrate that WT CFTR is predominantly expressed in ciliated cells, and ⌬F508 CFTR pathogenesis in native tissues, like heterologous cells, reflects loss of normal protein processing.