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Cystic Fibrosis Methods and Protocols pp 143–157Cite as

Studies of the Molecular Basis for Cystic Fibrosis Using Purified Reconstituted CFTR Protein

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Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 70))

Abstract

Studies of purified, reconstituted cystic fibrosis transmembrane conductance regulator (CFTR) protein have proven invaluable in defining those functions that are intrinsic to the CFTR molecule. Reconstitution of purified CFTR protein in planar lipid bilayers provided direct evidence that CFTR possesses intrinsic activity as a protein kinase A (PKA)-regulated chloride channel (1). Further, it appears that the regulation of the CFTR channel gate (the structure essential for opening and closing of the channel pore) reported in biological membranes has been recapitulated with fidelity in studies of reconstituted purified protein. For example, patch clamp studies on biological membranes revealed that ATP binding and/or hydrolysis by the PKA-phosphorylated CFTR channel caused channel opening and closing (2-6). This unique requirement for ATP in channel gating was also reported in reconstitution studies of the purified protein (7,8), indicating that the CFTR protein itself can mediate regulated chloride flux without the requirement for accessory proteins. These results also justify the use of purified protein in detailed biochemical and structural studies of mechanisms underlying the function of this protein. The CFTR molecule is known to be comprised of two membrane-spanning domains (TMDs), two nucleotide-binding domains (NBDs), and the phosphorylationdependent regulatory or R domain (9). However, to date, our understanding of the structure of these domains and the coordination of their activities is limited.

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Author information

Authors and Affiliations

  1. Programme in Cell Biology, Research Institute, Hospital for Sick Children and the Department of Physiology, Faculty of Medicine, Toronto, Ontario, Canada

    Ilana Kogan, Mohabir Ramjeesingh, Canhui Li & Christine E. Bear

Authors
  1. Ilana Kogan
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  2. Mohabir Ramjeesingh
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  3. Canhui Li
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  4. Christine E. Bear
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Editor information

Editors and Affiliations

  1. Division of Molecular Medicine, Oregon Health Sciences University, Portland, OR

    William R. Skach MD

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© 2002 Humana Press Inc.

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Kogan, I., Ramjeesingh, M., Li, C., Bear, C.E. (2002). Studies of the Molecular Basis for Cystic Fibrosis Using Purified Reconstituted CFTR Protein. In: Skach, W.R. (eds) Cystic Fibrosis Methods and Protocols. Methods in Molecular Medicine™, vol 70. Humana Press. https://doi.org/10.1385/1-59259-187-6:143

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  • DOI: https://doi.org/10.1385/1-59259-187-6:143

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-897-4

  • Online ISBN: 978-1-59259-187-9

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