title: Multiple membrane-cytoplasmic domain contacts in the cystic fibrosis transmembrane conductance regulator (CFTR) mediate regulation of channel gating.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) ion channel mutated in patients with cystic fibrosis. The most common mutation, deletion of phenylalanine 508 (DeltaF508) and many other disease-associated mutations occur in the nucleotide binding domains (NBD) and the cytoplasmic loops (CL) of the membrane-spanning domains (MSD). A recently constructed computational model of the CFTR three-dimensional structure, supported by experimental data (Serohijos, A. W., Hegedus, T., Aleksandrov, A. A., He, L., Cui, L., Dokholyan, N. V., and Riordan, J. R. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 3256-3261) revealed that several of these mutations including DeltaF508 disrupted interfaces between these domains. Here we have used cysteine cross-linking experiments to verify all NBD/CL interfaces predicted by the structural model and observed that their cross-linking has a variety of different effects on channel gating. The interdomain contacts comprise aromatic clusters important for stabilization of the interfaces and also involve the Q-loops and X-loops that are in close proximity to the ATP binding sites. Cross-linking of all domain-swapping contacts between NBDs and MSD cytoplasmic loops in opposite halves of the protein rapidly and reversibly arrest single channel gating while those in the same halves have lesser impact. These results reinforce the idea that mediation of regulatory signals between cytoplasmic- and membrane-integrated domains of the CFTR channel apparently relies on an array of precise but highly dynamic interdomain structural joints.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Lihua
authors.initials: L
authors.lastname: He
authors.forename: Andrei A
authors.initials: AA
authors.lastname: Aleksandrov
authors.forename: Adrian W R
authors.initials: AW
authors.lastname: Serohijos
authors.forename: Tamás
authors.initials: T
authors.lastname: Hegedus
authors.forename: Luba A
authors.initials: LA
authors.lastname: Aleksandrov
authors.forename: Liying
authors.initials: L
authors.lastname: Cui
authors.forename: Nikolay V
authors.initials: NV
authors.lastname: Dokholyan
authors.forename: John R
authors.initials: JR
authors.lastname: Riordan
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics; D017354:Point Mutation; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator