title: Effect of deletion mutations on the function of CFTR chloride channels.
abstract: Mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a unique R domain; CFTR functions as a Cl- channel regulated by phosphorylation and by nucleoside triphosphates. To study the domains of CFTR involved in Cl- channel function, we expressed mutants lacking various domains and assayed cAMP-stimulated Cl- channel activity using the halide-sensitive fluorophore, 6-methoxy-N-(3'-sulfopropyl)-quinolinium. We previously reported that deletion of part of the R domain (residues 708-835) produced Cl- channels that were constitutively open. Here we show that more extensive deletions within the R domain failed to generate functional CFTR Cl- channels; the portion of protein that could be deleted without destroying function corresponds to sequences that are not conserved in related proteins. In contrast, when we deleted the two NBDs (either alone, together, or in combination with the R domain), we did not observe functional Cl- channels. CFTR has a unique carboxyl terminus that is conserved across species. However, truncation of the carboxyl terminus (up to, but not including, NBD2) produced a regulated anion permeability similar to that of wild-type CFTR, suggesting that this region is not essential for channel function. Expression of two CF-associated nonsense mutants (G542X and W1316X) also failed to generate functional CFTR Cl- channels. These results help define structure:function relationships for CFTR and identify the domains that are required for Cl- channel function.
authors.affiliation: Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
authors.forename: D P
authors.initials: DP
authors.lastname: Rich
authors.forename: R J
authors.initials: RJ
authors.lastname: Gregory
authors.forename: S H
authors.initials: SH
authors.lastname: Cheng
authors.forename: A E
authors.initials: AE
authors.lastname: Smith
authors.forename: M J
authors.initials: MJ
authors.lastname: Welsh
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005656:Fungal Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; C505032:CFTR protein, human; D018118:Chloride Channels; D005656:Fungal Proteins; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator