A Simple Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles Armelle Varnier • Fre ´de ´rique Kermarrec • Iulia Blesneac • Christophe Moreau • Lavinia Liguori • Jean Luc Lenormand • Nathalie Picollet-D’hahan Received: 28 July 2009 / Accepted: 28 December 2009 / Published online: 5 February 2010 Ó Springer Science+Business Media, LLC 2010 Abstract A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the con- centrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from sub- micron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs. Keywords Biomembranes  Membrane protein  Membrane channel  Giant unilamellar vesicle  Proteoliposome  Fusion  Lipid Introduction Giant unilamellar vesicles (GUVs) and proteo-GUVs are models for biological membranes, with a size in the same range as that of cells (10–100 lm). In contrast to small unilamellar vesicles (SUVs, 30–50 nm) and large unila- mellar vesicles (LUVs, 100–200 nm), GUVs can be stud- ied with optical microscopic techniques such as phase contrast, differential interference contrast and fluorescence microscopy (Bagatolli 2006; Menger and Keiper 1998; Girard et al. 2004). In addition to imaging methods, the quantitative study of dynamics in membranes can be achieved with methods such as fluorescence recovery after photobleaching (FRAP) (Tareste et al. 2008), fluorescence correlation spectroscopy (FCS) (Kahya et al. 2003) and scanning fluorescence correlation spectroscopy (SFCS) (Ruan et al. 2004). GUVs can also be micromanipulated with pipettes (Sun et al. 2009) or with patch-clamp elec- trodes (Wunder and Colombini 1991), and recently flow- cytometric techniques were applied to these biomimetic systems (Lamblet et al. 2008). Different applications of GUVs have been reported, such as electrophysiological recordings of ion channels (Wunder and Colombini 1991; Regueiro et al. 1996; Kreir et al. 2008), studies of mem- brane–protein interactions (Ruan et al. 2004; Lee et al. 2008; Tamba and Yamazaki 2005; Riske and Do ¨bereiner 2003), DNA–membrane interactions (Angelova and Tso- neva 1999), lipid domain characteristics (Kahya et al. 2003; Bagatolli and Gratton 2000), actin-based motile processes (Delatour et al. 2008) and membrane morpho- logical changes induced by local pH gradient (Khalifat et al. 2008). GUVs have also been used as biomimetic containers that were fused by micromanipulation in order to study single-molecule enzyme activity (Hsin and Yeung 2007). A. Varnier  F. Kermarrec  N. Picollet-D’hahan (&) CEA, DSV, iRTSV, Biopuces, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France e-mail: Nathalie.picollet-dhahan@cea.fr I. Blesneac  C. Moreau IBS (CEA, CNRS, UJF), Prote ´ines Membranaires, 41 rue Jules Horowitz, 38027 Grenoble, France L. Liguori  J. L. Lenormand HumProTher Laboratory, TheREx, TIMC-IMAG, CNRS UMR5525, UJF, UFR de Me ´decine, 38706 La Tronche Cedex, France 123 J Membrane Biol (2010) 233:85–92 DOI 10.1007/s00232-010-9227-8