title: Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is an ATP-binding cassette transporter that contains conserved nucleotide-binding domains (NBDs). In CFTR, the NBDs bind and hydrolyze ATP to open and close the channel. Crystal structures of related NBDs suggest a structural model with an important signaling role for a gamma-phosphate linker peptide that couples bound nucleotide to movement of an alpha-helical subdomain. We mutated two residues in CFTR that the structural model predicts will uncouple effects of nucleotide binding from movement of the alpha-helical subdomain. These residues are Gln-493 and Gln-1291, which may directly connect the ATP gamma-phosphate to the gamma-phosphate linker, and residues Asn-505 and Asn-1303, which may form hydrogen bonds that stabilize the linker. In NBD1, Q493A reduced the frequency of channel opening, suggesting a role for this residue in coupling ATP binding to channel opening. In contrast, N505C increased the frequency of channel opening, consistent with a role for Asn-505 in stabilizing the inactive state of the NBD. In NBD2, Q1291A decreased the effects of pyrophosphate without altering other functions. Mutations of Asn-1303 decreased the rate of channel opening and closing, suggesting an important role for NBD2 in controlling channel burst duration. These findings are consistent with both the bacterial NBD structural model and gating models for CFTR. Our results extend models of nucleotide-induced structural changes from bacterial NBDs to a functional mammalian ATP-binding cassette transporter.
authors.affiliation: Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
authors.forename: Allan L
authors.initials: AL
authors.lastname: Berger
authors.forename: Mutsuhiro
authors.initials: M
authors.lastname: Ikuma
authors.forename: John F
authors.initials: JF
authors.lastname: Hunt
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Thomas
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Welsh
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010710:Phosphates / Q000737:chemistry*
chemical_list: D010710:Phosphates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000244:Adenosine Diphosphate; D001216:Asparagine; D000255:Adenosine Triphosphate; D005998:Glycine