Stabilizing rescued surface-localized δf508 CFTR by potentiation of its interaction with Na(+)/H(+) exchanger regulatory factor 1.

Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in CFTR, a plasma-membrane-localized anion channel. The most common mutation in CFTR, deletion of phenylalanine at residue 508 (ΔF508), causes misfolding of CFTR resulting in little or no protein at the plasma membrane. The CFTR corrector VX-809 shows promise for treating CF patients homozygous for ΔF508. Here, we demonstrate the significance of protein-protein interactions in enhancing the stability of the ΔF508 CFTR mutant channel protein at the plasma membrane. We determined that VX-809 prolongs the stability of ΔF508 CFTR at the plasma membrane. Using competition-based assays, we demonstrated that ΔF508 CFTR interacts poorly with Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) compared to wild-type CFTR, and VX-809 significantly increased this binding affinity. We conclude that stabilized CFTR-NHERF1 interaction is a determinant of the functional efficiency of rescued ΔF508 CFTR. Our results demonstrate the importance of macromolecular-complex formation in stabilizing rescued mutant CFTR at the plasma membrane and suggest this to be foundational for the development of a new generation of effective CFTR-corrector-based therapeutics.

Functional regulation of cystic fibrosis transmembrane conductance regulator-containing macromolecular complexes: a small-molecule inhibitor approach.

CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to form multiple protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signalling in cells. Targeting protein-protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted the PDZ domain-based LPA2 (type 2 lysophosphatidic acid receptor)-NHERF2 (Na+/H+ exchanger regulatory factor-2) interaction within the CFTR-NHERF2-LPA2-containing macromolecular complexes in airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits the LPA2-NHERF2 interaction. We show that this compound can disrupt the LPA2-NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elevates cAMP levels in proximity to CFTR and upregulates its channel activity. The results of the present study demonstrate that CFTR Cl- channel function can be finely tuned by modulating PDZ domain-based protein-protein interactions within the CFTR-containing macromolecular complexes. The present study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl- channels.

Glucose transport by epithelia prepared from harvested enterocytes.

Transformed and cultured cell lines have significant shortcomings for investigating the characteristics and responses of native villus enterocytes in situ. Interpretations of results from intact tissues are complicated by the presence of underlying tissues and the crypt compartment. We describe a simple, novel, and reproducible method for preparing functional epithelia using differentiated enterocytes harvested from the small intestine upper villus of adult mice and preterm pigs with and without necrotizing enterocolitis. Concentrative, rheogenic glucose uptake was used as an indicator of epithelial function and was demonstrated by cellular accumulation of tracer (14)C D-glucose and Ussing chamber based short-circuit currents. Assessment of the epithelia by light and immunofluorescent microscopy revealed the harvested enterocytes remain differentiated and establish cell-cell connections to form polarized epithelia with distinct apical and basolateral domains. As with intact tissues, the epithelia exhibit glucose induced short-circuit currents that are increased by exposure to adenosine and adenosine 5'-monophosphate (AMP) and decreased by phloridzin to inhibit the apical glucose transporter SGLT-1. Similarly, accumulation of (14)C D-glucose by the epithelia was inhibited by phloridzin, but not phloretin, and was stimulated by pre-exposure to AMP and adenosine, apparently by a microtubule-based mechanism that is disrupted by nocodazole, with the magnitudes of responses to adenosine, forskolin, and health status exceeding those we have measured using intact tissues. Our findings indicate that epithelia prepared from harvested enterocytes provide an alternative approach for comparative studies of the characteristics of nutrient transport by the upper villus epithelium and the responses to different conditions and stimuli.

Compartmentalized cyclic adenosine 3',5'-monophosphate at the plasma membrane clusters PDE3A and cystic fibrosis transmembrane conductance regulator into microdomains.

Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3',5'-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function. Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex. Consequently, compartmentalized cAMP signaling is lost. PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. The physiological relevance of PDE3A-CFTR interaction was investigated using pig trachea submucosal gland secretion model. Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.

Clinical and molecular characterization of S1118F-CFTR.

BACKGROUND: Cystic fibrosis is a lethal autosomal recessive disorder usually associated with lung disease, pancreatic insufficiency and high sweat chloride levels. CLINICAL CASE: A patient admitted to Le Bonheur Children's Medical Center (LBCMC, Memphis, TN) showed symptoms of meconium ileus which required exploratory laparotomy, bowel resection and ileostomy. Genotyping showed DeltaF508/I1027T on one chromosome and S1118F on the other. Sweat testing on three different occasions gave negative and intermediate results (22.7, 24.6 mmol/L; 55.1, 58.6 mmol/L and 55.1, 58 mmol/L) and pancreatic elastase testing showed normal levels. OBJECTIVE: To characterize S1118F-CFTR mutation at a molecular level to help understand the associated CF-phenotype. METHODS: Molecular characterization of S1118F-CFTR mutant was studied in HEK-293 cells at 37 degrees C. Various biochemical methods such as Western blotting, real-time PCR, Pulse chase labeling and iodide efflux assay were employed. RESULTS: S1118F-CFTR makes less than 10-15% of mature CFTR (band C) compared to WT-CFTR. The mRNA levels of S1118F-CFTR and WT-CFTR are comparable. S1118F-CFTR is functional but shows about 10-15% of WT-CFTR activity. S1118F-CFTR shows impaired maturation and CF-correctors can increase the amount of mature and functional CFTR by three- to fourfold. CONCLUSION: S1118F-CFTR shows impaired maturation and an individual with S1118F-CFTR paired with DeltaF508-CFTR exhibits atypical CF symptoms with intermediate sweat chloride level and meconium ileus despite documented pancreatic sufficiency.

Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.