THE NUCLEOTIDE-BINDING FOLDS OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR ARE EXTRACELLULARLY ACCESSIBLE

Analysis of the primary sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) has suggested the presence of two predict ed cytoplasmic regions of the protein which are thought to be nucleoti de binding folds (NBF1 and NBF2). Previous studies have shown that pur ified recombinant NBF1 can form anion conducting channels in planar ph ospholipid bilayers [Arispe et al. (1992) Proc. Natl. Acad. Sci. U.S.A . 89, 1539-1543] and that the bacterial His P protein (analogous to a NBF) can be extracellularly labeled with a membrane-impermeant reagent [Baichwal et al. (1993) Proc, Natl. Acad. Sci. U.S.A. 90, 620-624], B ased on these observations, it is reasonable to hypothesize that the N BFs from the CFTR are associated with the plasma membrane and have ext racellularly-accessible regions. Direct biochemical evidence for this was obtained by determining the ability of the individual NBFs, expres sed in intact Hi5 cells, to be chemically modified with the membrane-i mpermeant reagent NHS-biotin, The results indicate that both NBF1 and NBF2, in intact cells, can be chemically modified by extracellular NHS -biotin. The negative control, the cytosolic enzyme beta-galactosidase , was not significantly labeled under these conditions, verifying the extracellular nature of the labeling reaction. When the surface-access ibility of a NBF1 construct containing the CF-causing mutation Delta F 508 was analyzed, similar labeling was observed, suggesting that the m utation does not affect this aspect of the CFTR's structure. These dat a support the conclusion that, under certain conditions, the NBFs are capable of spanning the plasma membrane, perhaps constituting a portio n of the CFTR's ion conducting channel.