title: Potentiation of disease-associated cystic fibrosis transmembrane conductance regulator mutants by hydrolyzable ATP analogs.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and DeltaF508. Previously we found that the ATP analog N(6)-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by approximately 7-fold. Here we show that 2'-deoxy-ATP (dATP), but not 3'-deoxy-ATP, increases the activity of G551D-CFTR by approximately 8-fold. We custom synthesized N(6)-(2-phenylethyl)-2'-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 +/- 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of DeltaF508-CFTR by increasing its activity by 19.5 +/- 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with DeltaF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable.
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri, 134 Research Park Dr., Columbia, MO 65211, USA.
authors.forename: Haruna
authors.initials: H
authors.lastname: Miki
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhou
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Tzyh-Chang
authors.initials: TC
authors.lastname: Hwang
authors.forename: Silvia G
authors.initials: SG
authors.lastname: Bompadre
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000502:physiology*; D004357:Drug Synergism; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D009154:Mutation; D018408:Patch-Clamp Techniques; D014162:Transfection
chemical_list: C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000255:Adenosine Triphosphate; D019833:Genistein
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