title: Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain.
abstract: ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.
authors.affiliation: Department of Internal Medicine, Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Allan L
authors.initials: AL
authors.lastname: Berger
authors.forename: Mutsuhiro
authors.initials: M
authors.lastname: Ikuma
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Welsh
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000502:physiology*; D019281:Dimerization; D006801:Humans; D006868:Hydrolysis; D015640:Ion Channel Gating
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate