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Conformational change of the extracellular parts of the CFTR protein during channel gating

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Abstract

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd2+ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.

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Abbreviations

ABC:

ATP-binding cassette

CF:

Cystic fibrosis

CFTR:

CF transmembrane conductance regulator

CHO:

Chinese hamster ovary

cryo-EM:

Cryo-electron microscopy

CuPhe:

Copper(II)-o-phenanthroline

DTT:

Dithiothreitol

ECL:

Extracellular loop

NBD:

Nucleotide-binding domain

TM:

Transmembrane helix

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Acknowledgements

We would like to thank Christina Irving for technical assistance. This work was supported by Cystic Fibrosis Canada.

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Authors and Affiliations

  1. Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada

    Alexander Negoda, Elizabeth A. Cowley, Yassine El Hiani & Paul Linsdell

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  1. Alexander Negoda
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  2. Elizabeth A. Cowley
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  3. Yassine El Hiani
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  4. Paul Linsdell
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Correspondence to Paul Linsdell.

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Negoda, A., Cowley, E.A., El Hiani, Y. et al. Conformational change of the extracellular parts of the CFTR protein during channel gating. Cell. Mol. Life Sci. 75, 3027–3038 (2018). https://doi.org/10.1007/s00018-018-2777-0

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  • DOI: https://doi.org/10.1007/s00018-018-2777-0

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