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J. Biol. Chem., Vol. 283, Issue 44, 30300-30310, October 31, 2008

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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^*

Kazuhiro Mio, Toshihiko Ogura, Muneyo Mio, Hiroyasu Shimizu^¶||, Tzyh-Chang Hwang^¶, Chikara Sato¹, and Yoshiro Sohma^¶**2

From the Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Umezono 1-1-4, Tsukuba, Ibaraki 305-8568, Japan, Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan, ^¶John M. Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211, Departments of ^||Hygiene and Public Health and ^**Physiology, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan, and Department of Pharmacology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan

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 162Å. 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.

Received for publication, April 25, 2008 , and in revised form, August 5, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants R01 DK55835-09 and R01 HL53445-11 (to T.-C. H.). This work was also supported by a grant-in-aid for scientific research on priority areas, structure of biological macromolecular assemblies (to K. M. and C. S.); by a grant from the Japan New Energy and Industrial Technology Development Organization (NEDO) (to C. S.); by a grant from PRESTO of the Japan Science and Technology Agency (to T. O.); and by Japan Society for the Promotion of Science (JSPS) Grant 19590215 (to Y. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. Tel.: 81-29-861-5562; Fax: 81-29-861-6478; E-mail: ti-sato{at}aist.go.jp.

² To whom correspondence may be addressed: Dept. of Pharmacology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: 81-3-5363-3750; Fax: 81-3-3359-8889; E-mail: yoshiros{at}sc.itc.keio.ac.jp.

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