title: Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.
abstract: The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.
authors.affiliation: From the Stead Family Department of Pediatrics.
authors.forename: Qian
authors.initials: Q
authors.lastname: Dong
authors.affiliation: the Department of Internal Medicine, the Howard Hughes Medical Institute, Iowa City, Iowa 52242.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Ernst
authors.affiliation: the Department of Internal Medicine.
authors.forename: Lynda S
authors.initials: LS
authors.lastname: Ostedgaard
authors.affiliation: the Department of Molecular Physiology and Biophysics, and the Medical Scientist Training Program, University of Iowa, Iowa City, Iowa 52242 and.
authors.forename: Viral S
authors.initials: VS
authors.lastname: Shah
authors.affiliation: From the Stead Family Department of Pediatrics.
authors.forename: Amanda R
authors.initials: AR
authors.lastname: Ver Heul
authors.affiliation: the Department of Internal Medicine, the Howard Hughes Medical Institute, Iowa City, Iowa 52242 the Department of Molecular Physiology and Biophysics, and.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Welsh
authors.affiliation: From the Stead Family Department of Pediatrics, christoph-randak@uiowa.edu.
authors.forename: Christoph O
authors.initials: CO
authors.lastname: Randak
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000249:Adenosine Monophosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000263:Adenylate Kinase / Q000235:genetics* / Q000378:metabolism; D020816:Amino Acid Motifs; D001665:Binding Sites; D019921:Biotinylation; D001980:Bronchi / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005973:Glutamine / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D011485:Protein Binding
chemical_list: D018528:ATP-Binding Cassette Transporters; C505032:CFTR protein, human; D018118:Chloride Channels; D005973:Glutamine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase
keywords: ABC transporter; AMP; ATP; adenylate kinase (ADK); chloride channel; cystic fibrosis; cystic fibrosis transmembrane conductance regulator (CFTR); epithelium; patch clamp; photoaffinity labeling