Reconstitution of Proteoliposomes for Phospholipid Scrambling and Nonselective Channel Assays

  • Maria E. Falzone
  • Alessio AccardiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2127)


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.

Key words

Phospholipids Scrambling Scramblases Lipid transport TMEM16 Nonselective channel 



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.


  1. 1.
    Pomorski T, Menon AK (2006) Lipid flippases and their biological functions. Cell Mol Life Sci 63(24):2908–2921CrossRefGoogle Scholar
  2. 2.
    Bevers EM, Williamson PL (2016) Getting to the outer leaflet: physiology of Phosphatidylserine exposure at the plasma membrane. Physiol Rev 96(2):605–645. Scholar
  3. 3.
    Kodigepalli KM, Bowers K, Sharp A, Nanjundan M (2015) Roles and regulation of phospholipid scramblases. FEBS Lett 589(1):3–14. Scholar
  4. 4.
    Nagata S, Suzuki J, Segawa K, Fujii T (2016) Exposure of phosphatidylserine on the cell surface. Cell Death Differ 23(6):952–961. Scholar
  5. 5.
    Suzuki J, Umeda M, Sims PJ, Nagata S (2010) Calcium-dependent phospholipid scrambling by TMEM16F. Nature 468:834–838CrossRefGoogle Scholar
  6. 6.
    Suzuki J, Fujii T, Imao T, Ishihara K, Kuba H, Nagata S (2013) Calcium-dependent phospholipid scramblase activity of TMEM16 protein family members. J Biol Chem 288(19):13305–13316. Scholar
  7. 7.
    Malvezzi M, Chalat M, Janjusevic R, Picollo A, Terashima H, Menon AK, Accardi A (2013) Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel. Nat Commun 4.
  8. 8.
    Brunner JD, Lim NK, Schenck S, Duerst A, Dutzler R (2014) X-ray structure of a calcium-activated TMEM16 lipid scramblase. Nature 516(7530):207–212CrossRefGoogle Scholar
  9. 9.
    Bushell SR, Pike ACW, Falzone ME, Rorsman NJG, Ta CM, Corey RA, Newport TD, Shintre CA, Tessitore A, Chu A, Wang Q, Shrestha L, Mukhopadhyay S, Love JD, Burgess-Brown NA, Sitsapesan R, Stansfeld PJ, Huiskonen JT, Tommaro P, Accardi A, Carpenter EP (2019) The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K. Nat Commun 10:3956. Scholar
  10. 10.
    Alvadia C, Lim NK, Clerico Mosina V, Oostergetel GT, Dutzler R, Paulino C (2019) Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F. Elife 8:e44365. Scholar
  11. 11.
    Di Zanni E, Gradogna A, Scholz-Starke J, Boccaccio A (2017) Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia. Cell Mol Life Sci 75:1–14. Scholar
  12. 12.
    Suzuki J, Denning DP, Imanishi E, Horvitz HR, Nagata S (2013) Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science 341(6144):403–406CrossRefGoogle Scholar
  13. 13.
    Suzuki J, Imanishi E, Nagata S (2016) Xkr8 phospholipid scrambling complex in apoptotic phosphatidylserine exposure. Proc Natl Acad Sci U S A 113(34):9509–9514CrossRefGoogle Scholar
  14. 14.
    Menon I, Huber T, Sanyal S, Banerjee S, Barré P, Canis S, Warren JD, Hwa J, Sakmar TP, Menon AK (2011) Opsin is a phospholipid flippase. Curr Biol 21(2):149–153. Scholar
  15. 15.
    Goren MA, Morizumi T, Menon I, Joseph JS, Dittman JS, Cherezov V, Stevens RC, Ernst OP, Menon AK (2014) Constitutive phospholipid scramblase activity of a G protein-coupled receptor. Nat Commun 5:5115. Scholar
  16. 16.
    Verchère A, Ou W-L, Ploier B, Morizumi T, Goren MA, Bütikofer P, Ernst OP, Khelashvili G, Menon AK (2017) Light-independent phospholipid scramblase activity of bacteriorhodopsin from Halobacterium salinarum. Sci Rep 7(1):9522–9522. Scholar
  17. 17.
    Kol MA, Van Laak ANC, Rijkers DTS, Antoinette Killian J, De Kroon AIPM, De Kruijff B (2003) Phospholipid flop induced by transmembrane peptides in model membranes is modulated by lipid composition. Biochemistry 42(1):231–237. Scholar
  18. 18.
    Mihajlovic M, Lazaridis T (2010) Antimicrobial peptides in Toroidal and cylindrical pores. Biochim Biophys Acta 1798(8):1485–1493. Scholar
  19. 19.
    Ohmann A, Li C-Y, Maffeo C, Al Nahas K, Baumann KN, Göpfrich K, Yoo J, Keyser UF, Aksimentiev A (2018) A synthetic enzyme built from DNA flips 10(7) lipids per second in biological membranes. Nat Commun 9(1):2426–2426. Scholar
  20. 20.
    Yu K, Whitlock JM, Lee K, Ea O, Yuan Cui Y, Hartzell HC (2015) Identification of a lipid scrambling domain in ANO6/TMEM16F. Elife 4:1–23. Scholar
  21. 21.
    Suzuki J, Imanishi E, Nagata S (2014) Exposure of phosphatidylserine by Xk-related protein family members during apoptosis. J Biol Chem 289(44):30257–30267CrossRefGoogle Scholar
  22. 22.
    Yang H, Kim A, David T, Palmer D, Jin T, Tien J, Huang F, Cheng T, Coughlin SR, Jan YN, Jan LY (2012) TMEM16F forms a Ca(2+)-activated Cation Channel required for lipid scrambling in platelets during blood coagulation. Cell 151(1):111–122CrossRefGoogle Scholar
  23. 23.
    Malvezzi M, Andra KK, Pandey K, Lee B-C, Brown A, Iqbal R, Menon AK, Accardi A (2018) Out of the groove transport of lipids by TMEM16 and GPCR scramblases PNAS. doi:
  24. 24.
    Lee B-C, Khelashvili G, Falzone M, Menon AK, Weinstein H, Accardi A (2018) Gating mechanism of the extracellular entry to the lipid pathway in a TMEM16 scramblase. Nat Commun 9(1):3251. Scholar
  25. 25.
    Falzone ME, Rheinberger J, Lee B-C, Peyear T, Sasset L, Raczkowski AM, Eng ET, Di Lorenzo A, Andersen OS, Nimigean CM, Accardi A (2019) Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase. Elife 8:e43229. doi:
  26. 26.
    Andra KK, Dorsey S, Royer C, Menon AK (2018) Structural mapping of fluorescently-tagged, functional nhTMEM16 scramblase in a lipid bilayer. J Biol Chem 293:12248. Scholar
  27. 27.
    Marx U, Lassmann G, Holzhutter HG, Wustner D, Muller P, Hohlig A, Kubelt J, Herrmann A (2000) Rapid flip-flop of phospholipids in endoplasmic reticulum membranes studied by a stopped-flow approach. Biophys J 78(5):2628–2640. Scholar
  28. 28.
    Sanyal S, Frank CG, Menon AK (2008) Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum. Biochemistry 47:7937–7946CrossRefGoogle Scholar
  29. 29.
    Sanyal S, Menon AK (2009) Specific transbilayer translocation of dolichol-linked oligosaccharides by an endoplasmic reticulum flippase. Proc Natl Acad Sci U S A 106(3):767–772CrossRefGoogle Scholar
  30. 30.
    Sanyal S, Menon AK (2010) Stereoselective transbilayer translocation of mannosyl phosphoryl dolichol by an endoplasmic reticulum flippase. Proc Natl Acad Sci U S A 107:11289–11294CrossRefGoogle Scholar
  31. 31.
    Wang L, Iwasaki Y, Andra KK, Pandey K, Menon AK, Bütikofer P (2018) Scrambling of natural and fluorescently tagged phosphatidylinositol by reconstituted G protein-coupled receptor and TMEM16 scramblases. J Biol Chem 293(47):18318–18327. Scholar
  32. 32.
    Lee B-C, Menon AK, Accardi A (2016) The nhTMEM16 Scramblase is also a nonselective Ion Channel. Biophys J 111(9):1919–1924.
  33. 33.
    Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, Pfeffer U, Ravazzolo R, Zegarra-Moran O, Galietta LJV (2008) TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science 322(5901):590–594. Scholar
  34. 34.
    Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim W-S, Park SP, Lee J, Lee B, Kim B-M, Raouf R, Shin YK, Oh U (2008) TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455(7217):1210–1215. Scholar
  35. 35.
    Schroeder BC, Cheng T, Jan YN, Jan LY (2008) Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134(6):11CrossRefGoogle Scholar
  36. 36.
    Whitlock JM, Hartzell HC (2016) A pore idea: the ion conduction pathway of TMEM16/ANO proteins is composed partly of lipid. Pflugers Arch 468(3):455–473. Scholar
  37. 37.
    Jiang T, Yu K, Hartzell HC, Tajkhorshid E (2017) Lipids and ions traverse the membrane by the same physical pathway in the. Elife 16:1–26. Scholar
  38. 38.
    Paulino C, Neldner Y, Lam AKM, Kalienkova V, Brunner JD, Schenck S, Dutzler R (2017) Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A. Elife 6:1–23. Scholar
  39. 39.
    Paulino C, Kalienkova V, Lam AKM, Neldner Y, Dutzler R (2017) Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM. Nature 552(7685):421–425. Scholar
  40. 40.
    Dang S, Feng S, Tien J, Peters CJ, Bulkley D, Lolicato M, Zhao J, Zuberbühler K, Ye W, Qi L, Chen T, Craik CS, Nung Jan Y, Minor DL Jr, Cheng Y, Yeh Jan L (2017) Cryo-EM structures of the TMEM16A calcium-activated chloride channel. Nature 552:426. Scholar
  41. 41.
    Khelashvili G, Falzone ME, Cheng X, Lee B-C, Accardi A, Weinstein H (2019) Dynamic modulation of the lipid translocation groove generates a conductive ion channel in Ca2+-bound nhTMEM16. Nat Commun 10(1):4972.
  42. 42.
    Scudieri P, Caci E, Venturini A, Sondo E, Pianigiani G, Marchetti C, Ravazzolo R, Pagani F, Galietta LJV (2015) Ion channel and lipid scramblase activity associated with expression of TMEM16F/ANO6 isoforms. J Physiol 59317(593):3829–384817. Scholar
  43. 43.
    Ploier B, Caro LN, Morizumi T, Pandey K, Pearring JN, Goren MA, Finnemann SC, Graumann J, Arshavsky VY, Dittman JS, Ernst OP, Menon AK (2016) Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants. Nat Commun 7:12832. Scholar
  44. 44.
    Kalienkova V, Clerico Mosina V, Bryner L, Oostergetel GT, Dutzler R, Paulino C (2019) Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM. Elife 8:e44364. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of BiochemistryWeill Cornell Medical CollegeNew YorkUSA
  2. 2.Department of AnesthesiologyWeill Cornell Medical CollegeNew YorkUSA
  3. 3.Department of Physiology and BiophysicsWeill Cornell Medical CollegeNew YorkUSA

Personalised recommendations