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

Qian Dong; Sarah E Ernst; Lynda S Ostedgaard; Viral S Shah; Amanda R Ver Heul; Michael J Welsh; Christoph O Randak

doi:10.1074/jbc.M114.611616

Back

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

Journal article

Open access

Peer reviewed

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

Qian Dong, Sarah E Ernst, Lynda S Ostedgaard, Viral S Shah, Amanda R Ver Heul, Michael J Welsh and Christoph O Randak

The Journal of biological chemistry, Vol.290(22), pp.14140-14153

05/29/2015

DOI: 10.1074/jbc.M114.611616

PMCID: PMC4447984

PMID: 25887396

Files and links (1)

url

https://doi.org/10.1074/jbc.M114.611616View

Published (Version of record) Open Access

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.

Immunohistochemistry

Biotinylation

Epithelial Cells - metabolism

Mutagenesis, Site-Directed

Humans

Adenosine Monophosphate - chemistry

Adenosine Diphosphate - chemistry

Cystic Fibrosis Transmembrane Conductance Regulator - metabolism

Amino Acid Motifs

Chloride Channels - metabolism

Gene Expression Regulation, Enzymologic

Patch-Clamp Techniques

Adenylate Kinase - metabolism

ATP-Binding Cassette Transporters - metabolism

Bronchi - metabolism

Protein Binding

Glutamine - chemistry

HeLa Cells

Mutation

Adenosine Triphosphate - chemistry

Adenylate Kinase - genetics

Binding Sites

Details

Title: Subtitle: 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
Creators: Qian Dong - From the Stead Family Department of Pediatrics
Sarah E Ernst - the Department of Internal Medicine, the Howard Hughes Medical Institute, Iowa City, Iowa 52242
Lynda S Ostedgaard - the Department of Internal Medicine
Viral S Shah - the Department of Molecular Physiology and Biophysics, and the Medical Scientist Training Program, University of Iowa, Iowa City, Iowa 52242 and
Amanda R Ver Heul - From the Stead Family Department of Pediatrics
Michael J Welsh - the Department of Internal Medicine, the Howard Hughes Medical Institute, Iowa City, Iowa 52242 the Department of Molecular Physiology and Biophysics, and
Christoph O Randak - From the Stead Family Department of Pediatrics, christoph-randak@uiowa.edu
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.290(22), pp.14140-14153
DOI: 10.1074/jbc.M114.611616
PMID: 25887396
PMCID: PMC4447984
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: P01 HL091842 / NHLBI NIH HHS Howard Hughes Medical Institute K08 HL097071 / NHLBI NIH HHS T32 GM007337 / NIGMS NIH HHS K08HL097071 / NHLBI NIH HHS HL091842 / NHLBI NIH HHS P01 HL051670 / NHLBI NIH HHS HL51670 / NHLBI NIH HHS P30 DK054759 / NIDDK NIH HHS
Language: English
Date published: 05/29/2015
Academic Unit: Neurology; Molecular Physiology and Biophysics; Pulmonary, Critical Care, and Occupational Medicine; Pulmonary Medicine; Stead Family Department of Pediatrics; Neurosurgery; Internal Medicine
Record Identifier: 9984020764602771

Metrics

16 Record Views

10 Times Cited - Web of Science