A CONSERVED REGION OF THE R-DOMAIN OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR IS IMPORTANT IN PROCESSING AND FUNCTION

The R domain of cystic fibrosis transmembrane conductance regulator (C FTR) connects the two halves of the protein, each of which possess a t ransmembrane-spanning domain and a nucleotide binding domain. Phosphor ylation of serine residues, which reside mostly within the C-terminal two-thirds of the R domain, is required for nucleotide-dependent activ ation of CFTR chloride channel activity. The N terminus of the R domai n is also likely to be important in CFTR function, since this region i s highly conserved among CFTRs of different species and exhibits seque nce similarity with the ''linker region'' of the related protein, P gl ycoprotein, To date, however, the role of this region in CFTR channel function remains unknown. In this paper, we report the effects of five disease-causing mutations within the N terminus of the CFTR-R domain. All five mutants exhibit defective protein processing in mammalian HE K-293 cells, suggesting that they are mislocalized and fail to reach t he cell surface. However, in the Xenopus oocyte, three mutants reached the plasma membrane. One of these mutants, L619S, exhibits no detecta ble function, whereas the other two, D614G and I618T, exhibit partial activity as chloride channels. Single channel analysis of these latter two mutants revealed that they possess defective rates of channel ope ning, consistent with the hypothesis that the N terminus of the R doma in participates in ATP-dependent channel gating, These findings suppor t recent structural models that include this region within extended bo undaries of the first nucleotide binding domain.