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

研究成果: Article査読

36 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)30300-30310
ページ数11
ジャーナルJournal of Biological Chemistry
283
44
DOI
出版ステータスPublished - 2008 10月 31
外部発表はい

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

フィンガープリント

「Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain」の研究トピックを掘り下げます。これらがまとまってユニークなフィンガープリントを構成します。

引用スタイル