CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) NUCLEOTIDE-BINDING DOMAIN-1 (NBD-1) AND CFTR TRUNCATED WITHIN NBD-1 TARGET TO THE EPITHELIAL PLASMA-MEMBRANE AND INCREASE ANION PERMEABILITY

The cystic fibrosis transmembrane conductance regulator (CFTR) is a me mber of the traffic ATPase family that includes multiple proteins char acterized by (1) ATP binding, (2) conserved transmembrane (TM) motifs and nucleotide binding domains (NBDs), and (3) molecular transport of small molecules across the cell membrane. While CFTR NBD-1 mediates AT P binding and hydrolysis, the membrane topology and function of this d omain in living eukaryotic cells remains uncertain. Tn these studies, we have expressed wild-type CFTR NBD-I (amino acids 433-586) or NBD-I containing the Delta F508 mutation transiently in COS-7 cells and esta blished that the domain is situated across the plasma membrane by four independent assays; namely, extracellular chymotrypsin digestion, sur face protein biotinylation, confocal immunofluorescent microscopy, and functional measurements of cell membrane anion permeability. Function al studies indicate that basal halide permeability is enhanced above c ontrol conditions following wild-type or Delta F508 NBD-1 expression i n three different epithelial cell lines. Furthermore, when clinically relevant CFTR proteins truncated within NBD-1 (R553X or G542X) are exp ressed, surface localization and enhanced halide permeability are agai n established. Together, these findings suggest that isolated CFTR NBD -I (with or without the Delta F508 mutation) is capable of targeting t he epithelial cell membrane and enhancing cellular halide permeability . Furthermore, CFTR truncated at position 553 or 542 and possessing th e majority of NBD-1 demonstrates surface localization and also confers increased halide permeability. These findings indicate that targeting to the plasma membrane and assumption of a transmembrane configuratio n are innate properties of the CFTR NBD-I. The results also support th e notion that components of the halide-selective pore of CFTR reside w ithin NBD-1.