The Second Half of the Cystic Fibrosis Transmembrane Conductance Regulator Forms a Functional Chloride Channel*
- From the Department of Physiology and Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) consists of two transmembrane domains (TMDs), TMD1 and TMD2, two cytoplasmic nucleotide binding domains (NBDs), NBD1 and NBD2, and a regulatory domain. To elucidate the complex function of the CFTR, deletion constructs encompassing the second half of the CFTR distal to the first transmembrane domain were expressed in Xenopusoocytes and IB3 cells (a cystic fibrosis cell line). Constructs containing the regulatory domain, the second transmembrane domain, and the second nucleotide binding domain formed constitutively active channels, which were further stimulated upon the addition of cAMP. On the other hand, a construct encompassing the second transmembrane domain and the second nucleotide binding domain was stimulated to a small but noticeable extent upon the addition of cAMP. The selectivity of the second-half construct was the same for iodide and chloride, in contrast to the selectivity of wild-type CFTR, which is Cl− > I−. However, both constructs displayed single-channel conductances that were significantly smaller than those displayed by the first half of the CFTR. We conclude that regions of the second transmembrane domain may contribute to the overall channel of the pore, although the first half of the CFTR may confer its selectivity.
Footnotes
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↵* This work was supported by the Cystic Fibrosis Foundation Research Development Program and National Institutes of Health Grants HL 51811, HL 47122, and DK 48977 (to W. B. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Physiology and Pediatrics, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-7166; Fax: 410-955-0461; E-mail: wguggino{at}bs.jhmi.edu.
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↵2 J. Cheng, personal communication.
- Abbreviations:
- CF
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cystic fibrosis
- CFTR
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CF transmembrane conductance regulator
- TMD
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transmembrane domain
- NBD
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nucleotide binding domain
- R
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regulatory
- wt
-
wild-type
- DIDS
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4,4′-diisothiocyanostilbene-2,2′-disulfonic acid.
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- Received August 3, 1998.
- Revision received August 25, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
