Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain

Kazuhiro Mio, Toshihiko Ogura, Muneyo Mio, Hiroyasu Shimizu, Tzyh Chang Hwang, Chikara Sato, Yoshiro Sohma

Research output: Contribution to journalArticle

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Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 x 106 x 162 Å. It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR·anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain.

Original languageEnglish
Pages (from-to)30300-30310
Number of pages11
JournalJournal of Biological Chemistry
Volume283
Issue number44
DOIs
Publication statusPublished - 2008 Oct 31

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Cystic Fibrosis Transmembrane Conductance Regulator
Chloride Channels
Orifices
Electron microscopy
Electron Microscopy
Size exclusion chromatography
Gel Chromatography
Observation
Blue Cross Blue Shield Insurance Plans
Native Polyacrylamide Gel Electrophoresis
Affinity chromatography
Wheat Germ Agglutinins
Proteins
Three-Dimensional Imaging
Antibodies
Saline water
HEK293 Cells
Domes
Protein Transport
Regulator Genes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain. / Mio, Kazuhiro; Ogura, Toshihiko; Mio, Muneyo; Shimizu, Hiroyasu; Hwang, Tzyh Chang; Sato, Chikara; Sohma, Yoshiro.

In: Journal of Biological Chemistry, Vol. 283, No. 44, 31.10.2008, p. 30300-30310.

Research output: Contribution to journalArticle

Mio, Kazuhiro ; Ogura, Toshihiko ; Mio, Muneyo ; Shimizu, Hiroyasu ; Hwang, Tzyh Chang ; Sato, Chikara ; Sohma, Yoshiro. / Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 44. pp. 30300-30310.
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