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.

Delivering CFTR to ciliated cells of cystic fibrosis (CF) patients fully restores ion and fluid transport to the lumenal surface of airway epithelium and returns mucus transport rates to those of non-CF airways.

After phosphorylation by protein kinase A, gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by the interaction of ATP with its nucleotide binding domains (NBDs). Models of this gating regulation have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet there has been little biochemical confirmation of the predictions of these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater role of NBD1 than NBD2. Separation of the domains by limited trypsin digestion and enrichment by immunoprecipitation confirmed greater and more stable nucleotide trapping at NBD1. This asymmetry of the two domains in interactions with nucleotides was reflected most emphatically in the response to the nonhydrolyzable ATP analogue, 5 -adenylyl-␤,␥-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing. Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2. This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at NBD2 and thereby questions the relevance of hydrolysis at that domain to channel closing.The cystic fibrosis transmembrane conductance regulator (CFTR) 1 has two nucleotide binding domains (NBDs) that are believed in some manner to regulate permeation of chloride ions (1). Whereas the two NBDs of another ABC protein, the P-glycoprotein multidrug transporter, are highly homologous and believed to be functionally equivalent (2), the domains in members of the ABCC subfamily to which CFTR belongs show less sequence similarity (3). This asymmetry is reflected functionally in two members of this subfamily, SUR1 (4, 5) and MRP1 (6 -8). Both ATP and ADP act on SUR1 to regulate the Kir6.2 potassium channel; ATP is bound at NBD1 and also bound and hydrolyzed at NBD2, and the ADP produced stabilizing ATP binding to NBD1 (9). Similarly in the case of MRP1, which transports conjugated anions, ATP binding was detected exclusively at NBD1 and was enhanced allosterically by the trapping of ADP produced by hydrolysis at NBD2 (6).Schemes of ATP binding and hydrolysis by CFTR have been proposed solely on the basis of channel-gating responses (10 -12). Implicit in these models is the idea that channel opening is driven by ATP binding and hydrolysis at one NBD and closing at the other (13). Intact CFTR (14) and bacterial fusion proteins containing either of the NBDs (15, 16) hydrolyze ATP. It has been reported also that photoaffinity labeling of CFTR by 8-azido-ATP at NBD1 occurred...

. ClC-3A resides in late endosomes where it serves as an anion shunt during acidification. We show here that the ClC-3B PDZ-binding isoform resides in the Golgi where it co-localizes with a small amount of the other known PDZ-binding chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator). Both channel proteins bind the Golgi PDZ protein, GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). Interestingly, however, when overexpressed, GOPC, which is thought to influence traffic in the endocytic/secretory pathway, causes a large reduction in the amounts of both channels, probably by leading them to the degradative end of this pathway. ClC-3B as well as CFTR also binds EBP50 (ERM-binding phosphoprotein 50) and PDZK1, which are concentrated at the plasma membrane. However, only PDZK1 was found to promote interaction between the two channels, perhaps because they were able to bind to two different PDZ domains in PDZK1. Thus while small portions of the populations of ClC-3B and CFTR may associate and co-localize, the bulk of the two populations reside in different organelles of cells where they are expressed heterologously or endogenously, and therefore their cellular functions are likely to be distinct and not primarily related.

Many cystic fibrosis transmembrane conductance regulator (CFTR) mutants are recognized as aberrant by the quality control apparatus at the endoplasmic reticulum (ER) and are targeted for degradation. The mechanism whereby nascent chains are distinguished as either competent or incompetent for ER export has not been elucidated. Here we show that export-incompetent chains display multiple arginine-framed tripeptide sequences like the one recently identified in ATP-sensitive K+ channels. Replacement of arginine residues at positions R29, R516, R555, and R766 with lysine residues to inactivate four of these motifs simultaneously causes delta F508 CFTR, present in approximately 90% of CF patients, to escape ER quality control and function at the cell surface. Interference with recognition of these signals may be helpful in the management of CF.