title: Converting nonhydrolyzable nucleotides to strong cystic fibrosis transmembrane conductance regulator (CFTR) agonists by gain of function (GOF) mutations.
abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) is the only ligand-gated ion channel that hydrolyzes its agonist, ATP. CFTR gating has been argued to be tightly coupled to its enzymatic activity, but channels do open occasionally in the absence of ATP and are reversibly activated (albeit weakly) by nonhydrolyzable nucleotides. Why the latter only weakly activates CFTR is not understood. Here we show that CFTR activation by adenosine 5'-O-(thiotriphosphate) (ATPγS), adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP), and guanosine 5'-3-O-(thio)triphosphate (GTPγS) is enhanced substantially by gain of function (GOF) mutations in the cytosolic loops that increase unliganded activity. This enhancement correlated with the base-line nucleotide-independent activity for several GOF mutations. AMP-PNP or ATPγS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). GOF mutant channels deactivated very slowly upon AMP-PNP or ATPγS removal (τdeac ∼ 100 s) implying tight binding between the two NBDs. Despite this apparently tight binding, neither AMP-PNP nor ATPγS activated even the strongest GOF mutant as strongly as ATP. ATPγS-activated wild type channels deactivated more rapidly, indicating that GOF mutations in the cytosolic loops reciprocally/allosterically affect nucleotide occupancy of the NBDs. A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPγS, indicating that these analogs interact differently with the NBDs. We conclude that poorly hydrolyzable nucleotides are less effective than ATP at opening CFTR channels even when they bind tightly to the NBDs but are converted to stronger agonists by GOF mutations.
authors.affiliation: Department of Cell, Developmental, and Integrative Biology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
authors.forename: George
authors.initials: G
authors.lastname: Okeyo
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Shipeng
authors.initials: S
authors.lastname: Wei
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Kirk
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology*; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000819:agonists* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D015640:Ion Channel Gating; D008564:Membrane Potentials; D020125:Mutation, Missense; D018408:Patch-Clamp Techniques; D054730:Protein Interaction Domains and Motifs
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate
keywords: ABC Transporter; ATP; ATPases; Allosteric Regulation; CFTR; Cystic Fibrosis; Ion Channels; Ligand; Ligand-binding Protein