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.

PDE3A functionally and physically interacts with CFTR. Inhibition of PDE3A generates compartmentalized cAMP, which further clusters PDE3A and CFTR into microdomains at the plasma membrane of epithelial cells and potentiates CFTR channel function. Our findings provide insights into the important role of PDE3A in compartmentalized cAMP signaling.

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.

SYNOPSIS CFTR has been shown to form multiple-protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signaling in cells. Targeting protein-protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted PDZ-based LPA2-NHERF2 interaction within the CFTR-NHERF2-LPA2-containing macromolecular complexes at airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits LPA2-NHERF2 interaction. We show that this compound can disrupt 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. Our results demonstrate that CFTR Cl− channel function can be finely tuned by modulating PDZ-based protein-protein interactions within the CFTR-containing macromolecular complexes. Our study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl− channel.

compelling basic science and animal model observations would provide a solid foundation upon which to implement additional trials of "anti-coagulants" in IPF. In conclusion, we have answered the question of the utility of warfarin in progressive IPF, but many questions remain.