title: Reconstitution of a transmembrane protein, the voltage-gated ion channel, KvAP, into giant unilamellar vesicles for microscopy and patch clamp studies.
abstract: Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods - electroformation and gel-assisted swelling - to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.
authors: Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie.
authors: Matthias
authors: M
authors: Garten
authors: Kavli Institute for Brain and Mind, University of California, San Diego.
authors: Sophie
authors: S
authors: Aimon
authors: Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie; Patricia.Bassereau@curie.fr.
authors: Patricia
authors: P
authors: Bassereau
authors: Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health.
authors: Gilman E S
authors: GE
authors: Toombes
mesh_terms: D008856:Microscopy, Fluorescence / Q000379:methods; D018408:Patch-Clamp Techniques / Q000379:methods; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry* / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D024642:Potassium Channels, Voltage-Gated; D053835:Unilamellar Liposomes
references: Nat Rev Mol Cell Biol. 2009 Sep;10 (9):644-50
references: 19672276
references: Biochim Biophys Acta. 2012 Nov;1818(11):2598-604
references: 22652256
references: Biophys J. 2001 Sep;81(3):1464-74
references: 11509360
references: Proc Natl Acad Sci U S A. 2014 Apr 8;111(14):5083-7
references: 24706877
references: Curr Opin Cell Biol. 2008 Aug;20(4):476-82
references: 18539448
references: Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5622-6
references: 19304798
references: Science. 2011 Sep 2;333(6047):1252-4
references: 21885774
references: Biochim Biophys Acta. 2009 Oct;1788(10 ):2291-300
references: 19665445
references: Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4141-6
references: 20160074
references: J Am Chem Soc. 2009 Feb 11;131(5):1810-9
references: 19154115
references: Biophys J. 1981 Sep;35(3):637-52
references: 7272454
references: Nature. 2003 Mar 13;422(6928):180-5
references: 12629550
references: Biophys J. 2012 Feb 8;102(3):523-31
references: 22325275
references: Phys Rev Lett. 2005 Mar 4;94(8):088102
references: 15783939
references: Proc Natl Acad Sci U S A. 2011 Aug 2;108(31):12605-10
references: 21768336
references: Chembiochem. 2010 May 3;11(7):848-65
references: 20336703
references: BMC Cell Biol. 2010 Aug 12;11:65
references: 20704729
references: Phys Rev Lett. 2009 Jan 23;102(3):038102
references: 19257398
references: Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4697-702
references: 18353990
references: Biophys J. 2013 Mar 19;104(6):1248-56
references: 23528084
references: Biophys J. 1996 Oct;71(4):2087-93
references: 8889183
references: Biophys J. 2010 Mar 3;98(5):762-72
references: 20197029
references: Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19276-81
references: 19050073
references: Biophys J. 2009 Aug 5;97(3):738-47
references: 19651032
references: Dev Cell. 2014 Jan 27;28(2):212-8
references: 24480645
references: Biophys J. 2013 Jul 2;105(1):154-64
references: 23823234
references: Biophys J. 2005 Feb;88(2):1134-42
references: 15574707
references: Proc Natl Acad Sci U S A. 2013 Apr 30;110(18):7276-81
references: 23589883
references: PLoS One. 2011;6(10):e25529
references: 21998666
references: J Cell Physiol. 1969 Feb;73(1):49-60
references: 5765779
references: J Vis Exp. 2013 Jul 13;(77):e50436
references: 23892292
references: Methods Enzymol. 2009;465:161-76
references: 19913167
references: Nature. 2003 May 1;423(6935):33-41
references: 12721618
references: Biophys J. 1996 Mar;70(3):1112-21
references: 8785271