Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating

Allan L Berger; Mutsuhiro Ikuma; John F Hunt; Philip J Thomas; Michael J Welsh

doi:10.1074/jbc.M109539200

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Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating

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Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating

Allan L Berger, Mutsuhiro Ikuma, John F Hunt, Philip J Thomas and Michael J Welsh

The Journal of biological chemistry, Vol.277(3), pp.2125-2131

01/18/2002

DOI: 10.1074/jbc.M109539200

PMID: 11788611

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Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is an ATP-binding cassette transporter that contains conserved nucleotide-binding domains (NBDs). In CFTR, the NBDs bind and hydrolyze ATP to open and close the channel. Crystal structures of related NBDs suggest a structural model with an important signaling role for a gamma-phosphate linker peptide that couples bound nucleotide to movement of an alpha-helical subdomain. We mutated two residues in CFTR that the structural model predicts will uncouple effects of nucleotide binding from movement of the alpha-helical subdomain. These residues are Gln-493 and Gln-1291, which may directly connect the ATP gamma-phosphate to the gamma-phosphate linker, and residues Asn-505 and Asn-1303, which may form hydrogen bonds that stabilize the linker. In NBD1, Q493A reduced the frequency of channel opening, suggesting a role for this residue in coupling ATP binding to channel opening. In contrast, N505C increased the frequency of channel opening, consistent with a role for Asn-505 in stabilizing the inactive state of the NBD. In NBD2, Q1291A decreased the effects of pyrophosphate without altering other functions. Mutations of Asn-1303 decreased the rate of channel opening and closing, suggesting an important role for NBD2 in controlling channel burst duration. These findings are consistent with both the bacterial NBD structural model and gating models for CFTR. Our results extend models of nucleotide-induced structural changes from bacterial NBDs to a functional mammalian ATP-binding cassette transporter.

Glycine - genetics

Glycine - metabolism

Mutagenesis, Site-Directed

Phosphates - chemistry

Cystic Fibrosis Transmembrane Conductance Regulator - metabolism

Cystic Fibrosis Transmembrane Conductance Regulator - chemistry

Patch-Clamp Techniques

Adenosine Triphosphate - metabolism

Asparagine - metabolism

Cystic Fibrosis Transmembrane Conductance Regulator - genetics

Adenosine Diphosphate - metabolism

Ion Channel Gating

Amino Acid Substitution

Asparagine - genetics

Details

Title: Subtitle: Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating
Creators: Allan L Berger - Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA
Mutsuhiro Ikuma
John F Hunt
Philip J Thomas
Michael J Welsh
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.277(3), pp.2125-2131
DOI: 10.1074/jbc.M109539200
PMID: 11788611
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: HL29851 / NHLBI NIH HHS DK25295 / NIDDK NIH HHS HL51670 / NHLBI NIH HHS
Language: English
Date published: 01/18/2002
Academic Unit: Neurology; Molecular Physiology and Biophysics; Pulmonary, Critical Care, and Occupational Medicine; Neurosurgery; Internal Medicine
Record Identifier: 9984020622902771

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