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Reconstitution of Proteoliposomes for Phospholipid Scrambling and Nonselective Channel Assays

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Expression, Purification, and Structural Biology of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2127))

Abstract

Phospholipid scramblases catalyze the rapid trans-bilayer movement of lipids down their concentration gradients. This process is essential for numerous cellular signaling functions including cell fusion, blood coagulation, and apoptosis. The importance of scramblases is highlighted by the number of human diseases caused by mutations in these proteins. Because of their indispensable function, it is essential to understand and characterize the molecular function of phospholipid scramblases. Powerful tools to measure lipid transport in cells are available. However, these approaches provide limited mechanistic insights into the molecular bases of scrambling. Here we describe in detail an in vitro phospholipid scramblase assay and the accompanying analysis which allows for determination of the macroscopic rate constants associated with phospholipid scrambling. Notably, members of the TMEM16 family of scramblases also function as nonselective ion channels. To better understand the physiological relevance of this channel function as well as its relationship to the scrambling activity of the TMEM16s we also describe in detail an in vitro flux assay to measure nonselective channel activity. Together, these two assays can be used to investigate the dual activities of the TMEM16 scramblases/nonselective channels.

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Acknowledgement

The authors thank members of the Accardi lab for helpful discussions. This work was supported by NIH Grant R01GM106717 (to A.A.). M.E.F. is the recipient of a Weill Cornell Medicine Margaret & Herman Sokol Fellowship.

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Authors and Affiliations

  1. Department of Biochemistry, Weill Cornell Medical College, New York, NY, USA

    Maria E. Falzone & Alessio Accardi

  2. Department of Anesthesiology, Weill Cornell Medical College, New York, NY, USA

    Alessio Accardi

  3. Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA

    Alessio Accardi

Authors
  1. Maria E. Falzone
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  2. Alessio Accardi
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Correspondence to Alessio Accardi .

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Editors and Affiliations

  1. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Camilo Perez

  2. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Timm Maier

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Falzone, M.E., Accardi, A. (2020). Reconstitution of Proteoliposomes for Phospholipid Scrambling and Nonselective Channel Assays. In: Perez, C., Maier, T. (eds) Expression, Purification, and Structural Biology of Membrane Proteins. Methods in Molecular Biology, vol 2127. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0373-4_15

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  • DOI: https://doi.org/10.1007/978-1-0716-0373-4_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0372-7

  • Online ISBN: 978-1-0716-0373-4

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