title: A conserved region of the R domain of cystic fibrosis transmembrane conductance regulator is important in processing and function.
abstract: The R domain of cystic fibrosis transmembrane conductance regulator (CFTR) connects the two halves of the protein, each of which possess a transmembrane-spanning domain and a nucleotide binding domain. Phosphorylation of serine residues, which reside mostly within the C-terminal two-thirds of the R domain, is required for nucleotide-dependent activation of CFTR chloride channel activity. The N terminus of the R domain is also likely to be important in CFTR function, since this region is highly conserved among CFTRs of different species and exhibits sequence similarity with the "linker region" of the related protein, P-glycoprotein. 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 HEK-293 cells, suggesting that they are mislocalized and fail to reach the cell surface. However, in the Xenopus oocyte, three mutants reached the plasma membrane. One of these mutants, L619S, exhibits no detectable 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 opening, consistent with the hypothesis that the N terminus of the R domain participates in ATP-dependent channel gating. These findings support recent structural models that include this region within extended boundaries of the first nucleotide binding domain.
authors.affiliation: Division of Cell Biology, the Research Institute of the Hospital for Sick Children, Toronto M5G 1X8, Canada.
authors.forename: E A
authors.initials: EA
authors.lastname: Pasyk
authors.forename: X K
authors.initials: XK
authors.lastname: Morin
authors.forename: P
authors.initials: P
authors.lastname: Zeman
authors.forename: E
authors.initials: E
authors.lastname: Garami
authors.forename: K
authors.initials: K
authors.lastname: Galley
authors.forename: L J
authors.initials: LJ
authors.lastname: Huan
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: C E
authors.initials: CE
authors.lastname: Bear
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D017124:Conserved Sequence; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D005260:Female; D006801:Humans; D015640:Ion Channel Gating; D007668:Kidney; D008564:Membrane Potentials; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D010766:Phosphorylation; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine; D014162:Transfection; D014982:Xenopus laevis
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C505032:CFTR protein, human; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012694:Serine