The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a chloride channel protein that belongs to the superfamily of ATP binding cassette (ABC) transporters. Phosphorylation by protein kinase A in the presence of ATP activates the CFTR-mediated chloride conductance of the apical membranes. We have identified a novel hydrophilic CFTR binding protein, CAP70, which is also concentrated on the apical surfaces. CAP70 consists of four PDZ domains, three of which are capable of binding to the CFTR C terminus. Linking at least two CFTR molecules via cytoplasmic C-terminal binding by either multivalent CAP70 or a bivalent monoclonal antibody potentiates the CFTR chloride channel activity. Thus, the CFTR channel can be switched to a more active conducting state via a modification of intermolecular CFTR-CFTR contact that is enhanced by an accessory protein.

The most common defect in cystic fibrosis, deletion of phenylalanine from position 508 of the cystic fibrosis transmembrane conductance regulator (⌬F508 CFTR), decreases the trafficking of this protein to the cell surface membrane. Previous studies have shown that low temperature and high concentrations of glycerol or trimethylamine N-oxide can partially counteract the processing defect of ⌬F508 CFTR. The present study investigates whether physiologically relevant concentrations of organic solutes, accumulated by cotransporter proteins, can rescue the misprocessing of ⌬F508 CFTR. Myoinositol alone or myoinositol, betaine, and taurine given sequentially increased the processing of core-glycosylated, endoplasmic reticulum-arrested ⌬F508 CFTR into the fully glycosylated form of CFTR in IB3 cells or NIH 3T3 cells stably expressing ⌬F508 CFTR. Pulsechase experiments using transiently transfected COS7 cells demonstrated that organic solutes also increased the processing of the core-glycosylated form of green fluorescent protein-⌬F508 CFTR. Moreover, the prolonged half-life of the complex-glycosylated form of GFP-⌬F508 CFTR suggests that this treatment stabilized the mature form of the protein. In vitro studies of purified NBD1 stability and aggregation showed that myoinositol stabilized both the ⌬F508 and wild type CFTR and inhibited ⌬F508 misfolding. Most significantly, treatment of CF bronchial airway cells with these transportable organic solutes restores cAMP-stimulated single channel activity of both CFTR and outwardly rectifying chloride channel in the cell surface membrane and also restores a forskolin-stimulated macroscopic 36 Cl ؊ efflux. We conclude that organic solutes can repair CFTR functions by enhancing the processing of ⌬F508 CFTR to the plasma membrane by stabilizing the complex-glycosylated form of ⌬F508 CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) exhibits two conductance states, 9 picosiemens (pS) and 3 pS. To investigate the origin of these two distinct conductance states, we measured the single-channel activity of three truncated forms of CFTR. These include: TNR, which contains the first transmembrane domain, the first nucleotide binding domain, and the R domain; RT2N2, which contains the R domain, the second transmembrane domain, and the second nucleotide-binding domain; and T2N2, which contains only the second transmembrane domain and the second nucleotide-binding domain. The results show that TNR exhibits only the large conductance of 9.2 pS, whereas RT2N2 and T2N2 exhibit only the small conductance (3.8 -4.0 pS). Co-expression of TNR with T2N2 resulted in a mixed pattern of two conductance states, which is similar to that observed in wild-type CFTR. In further studies, a "dual-R mutant," R334W and R347P in the transmembrane segment 6 of the first half of CFTR, severely impaired the large conductance channel without affecting the small conductance channel. The ion selectivity and gating behavior of the two conductance channels are different regardless of whether they are measured in wild-type CFTR or in truncated CFTRs. The ion selectivity of the large conductance channel is Br ؊ > Cl ؊ > I ؊ , whereas the ion selectivity of the small conductance channel is BrThe open probability (P o ) of the large conductance is about 4-fold higher than that of the small conductance. Transition from closed to open states of the small conductance is not dependent upon the open or closed states of the large conductance. The independent behaviors of the two conductances in CFTR strongly suggest that CFTR may have two distinct pores. Thus, like ClC0, CFTR is likely to be a double-barreled ion channel, with the first half of CFTR forming the large conductance and the second half forming the small conductance. Quinton suggested in 1983 (1) that ClϪ transport is defective in cystic fibrosis and 6 years later, cloning of the cystic fibrosis gene (2) and subsequent studies showed that CFTR 1 is indeed a chloride channel (3-5). CFTR contains an anion selective pore with a linear I/V relationship (6, 7). In excised inside-out patches, wild-type CFTR exhibits two conductances, a main conductance of between 9 and 11 pS and a smaller conductance of approximately 4.0 pS. The selectivity of the main conductance is BrMany investigators have used mutagenesis to create CFTR cDNAs containing both naturally occurring and artificial mutations and truncated forms to CFTR to study the properties of the channel pore region. These studies as well as those using cysteine-scanning mutagenesis have pinpointed amino acid residues in the transmembrane segment 6 of TMD1 as critical to forming the main conductive pore of CFTR (3,4,8). We showed previously that TNR CFTR (comprising the first transmembrane domain, the first nucleotide binding domain, and the R domain) can form a functional chloride channel with characteristics approach...