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

• Published in: The Journal of biological chemistry Vol. 290; no. 22; pp. 14140 - 14153
• Main Authors: Dong, Qian, Ernst, Sarah E., Ostedgaard, Lynda S., Shah, Viral S., Ver Heul, Amanda R., Welsh, Michael J., Randak, Christoph O.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 29-05-2015; American Society for Biochemistry and Molecular Biology
• Subjects: ABC transporter; Adenosine Diphosphate - chemistry; Adenosine Monophosphate - chemistry; Adenosine Triphosphate - chemistry; adenylate kinase (ADK); Adenylate Kinase - genetics; Adenylate Kinase - metabolism; Amino Acid Motifs; AMP; ATP; ATP-Binding Cassette Transporters - metabolism; Binding Sites; Biotinylation; Bronchi - metabolism; chloride channel; Chloride Channels - metabolism; cystic fibrosis; cystic fibrosis transmembrane conductance regulator (CFTR); Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Enzymology; Epithelial Cells - metabolism; epithelium; Gene Expression Regulation, Enzymologic; Glutamine - chemistry; HeLa Cells; Humans; Immunohistochemistry; Mutagenesis, Site-Directed; Mutation; patch clamp; Patch-Clamp Techniques; photoaffinity labeling; Protein Binding; patch clamp; AMP; cystic fibrosis transmembrane conductance regulator (CFTR); chloride channel; adenylate kinase (ADK); cystic fibrosis; photoaffinity labeling; epithelium; ABC transporter; ATP
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.611616

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 P1,P5-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.
Background: Cystic fibrosis transmembrane conductance regulator (CFTR) channels have ATPase and adenylate kinase activity.
Results: Mutating Gln-1291 disrupts adenylate kinase- but not ATPase-dependent gating, and it reduces channel activity in airway epithelia.
Conclusion: Normal CFTR function in airway epithelia may depend on its adenylate kinase activity.
Significance: Adenylate kinase activity is important for the function of an ATP-binding cassette transporter.