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Functional and pharmacological induced structural changes of the cystic fibrosis transmembrane conductance regulator in the membrane solved using SAXS

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Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to the ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. To investigate the conformation of the CFTR in the membrane, we applied the small-angle x-ray scattering (SAXS) technique on microsomal membranes extracted from NIH/3T3 cells permanentely transfected with wild-type (WT) CFTR and with CFTR carrying the ΔF508 mutation. The electronic density profile of the membranes was calculated from the SAXS data, assuming the lipid bilayer electronic density to be composed by a series of Gaussian shells. The data indicate that membranes in the microsome vesicles, that contain mostly endoplasmic reticulum membranes, are oriented in the outside-out conformation. Phosphorylation does not change significantly the electronic density profile, while dephosphorylation produces a significant modification in the inner side of the profile. Thus, we conclude that the CFTR and its associated protein complex in microsomes are mostly phosphorylated. The electronic density profile of the ΔF508-CFTR microsomes is completely different from WT, suggesting a different assemblage of the proteins in the membranes. Low-temperature treatment of cells rescues the ΔF508-CFTR protein, resulting in a conformation that resembles the WT. Differently, treatment with the corrector VX-809 modifies the electronic profile of ΔF508-CFTR membrane, but does not recover completely the WT conformation. To our knowledge, this is the first report of a direct physical measurement of the structure of membranes containing CFTR in its native environment and in different functional and pharmacological conditions.

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Acknowledgements

This work was partially supported by the Fondazione per la Ricerca sulla Fibrosi Cistica, grant FFC#4/2012. These experiments were performed at the BL11-NCD beamline of ALBA Synchrotron Light Facility with financial support of the facility and the collaboration of ALBA staff. We thank Marc Malfois, Agneta Svenson and Christina Kamma-Lorger for the technical assistance at the synchrotron beamline. We are indebted to Dr. Cristina D’Arrigo for helping with the EM experiments. We thank also Alessandro Barbin for the construction of the sample cell for SAXS.

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  1. Istituto di Biofisica, CNR, via De Marini, 6, 16149, Genoa, Italy

    Debora Baroni & Oscar Moran

  2. Unità Operativa di Genetica Medica, Istituto G. Gaslini, Genoa, Italy

    Olga Zegarra-Moran

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  1. Debora Baroni
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Baroni, D., Zegarra-Moran, O. & Moran, O. Functional and pharmacological induced structural changes of the cystic fibrosis transmembrane conductance regulator in the membrane solved using SAXS. Cell. Mol. Life Sci. 72, 1363–1375 (2015). https://doi.org/10.1007/s00018-014-1747-4

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