title: Reconstitution of a Kv channel into lipid membranes for structural and functional studies.
abstract: To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas, USA.
authors.forename: Sungsoo
authors.initials: S
authors.lastname: Lee
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
authors.forename: Liang
authors.initials: L
authors.lastname: Shi
authors.forename: Qiu-Xing
authors.initials: QX
authors.lastname: Jiang
mesh_terms: D041522:Aeropyrum / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry*; D008823:Micelles; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D003902:Detergents; D008051:Lipid Bilayers; D008563:Membrane Lipids; D008823:Micelles; D024642:Potassium Channels, Voltage-Gated
references.citation: Annu Rev Biochem. 1998;67:199-225
references.pmid: 9759488
references.citation: Annu Rev Biophys Biomol Struct. 2004;33:269-95
references.pmid: 15139814
references.citation: Biochim Biophys Acta. 1990 Jun 27;1025(2):179-90
references.pmid: 2364077
references.citation: Chem Rev. 1997 Apr 1;97(2):391-410
references.pmid: 11848876
references.citation: Nature. 2003 Mar 13;422(6928):180-5
references.pmid: 12629550
references.citation: J Membr Biol. 1972;10(1):11-30
references.pmid: 4656230
references.citation: J Mol Biol. 2008 Sep 26;382(1):24-33
references.pmid: 18638484
references.citation: J Struct Biol. 2010 Mar;169(3):370-8
references.pmid: 19963066
references.citation: J Struct Biol. 2007 Feb;157(2):321-8
references.pmid: 16979348
references.citation: J Struct Biol. 2001 Jan;133(1):64-74
references.pmid: 11356065
references.citation: J Struct Biol. 2007 Dec;160(3):295-304
references.pmid: 17951070
references.citation: Q Rev Biophys. 1992 Feb;25(1):1-49
references.pmid: 1589568
references.citation: J Cell Biol. 1984 Mar;98(3):1054-62
references.pmid: 6699081
references.citation: Methods Enzymol. 2003;372:65-86
references.pmid: 14610807
references.citation: Biochim Biophys Acta. 2004 Nov 3;1666(1-2):62-87
references.pmid: 15519309
references.citation: Nat Commun. 2011;2:250
references.pmid: 21427721
references.citation: J Struct Biol. 1998;121(2):142-61
references.pmid: 9618341
references.citation: J Gen Physiol. 1980 Mar;75(3):251-70
references.pmid: 6247418
references.citation: Methods Mol Biol. 2009;504:291-321
references.pmid: 19159104
references.citation: Annu Rev Biophys Biomol Struct. 2003;32:257-83
references.pmid: 12543707
references.citation: Biochim Biophys Acta. 1992 Jun 30;1107(2):283-98
references.pmid: 1387003
references.citation: Nature. 2006 Dec 7;444(7120):775-9
references.pmid: 17136096
references.citation: Eur J Biochem. 1997 Feb 1;243(3):798-804
references.pmid: 9057848
references.citation: Curr Opin Struct Biol. 2012 Aug;22(4):529-36
references.pmid: 22483432
references.citation: Braz J Med Biol Res. 2002 Jul;35(7):753-66
references.pmid: 12131914
references.citation: Biophys J. 1994 May;66(5):1404-14
references.pmid: 8061192
references.citation: Biophys J. 1997 Jun;72(6):2545-58
references.pmid: 9168030
references.citation: J Mol Biol. 1968 Jan 28;31(2):227-35
references.pmid: 5635533
references.citation: Mol Biosyst. 2005 Sep;1(3):203-12
references.pmid: 16880984
references.citation: Nucleic Acids Res. 2012 Jul;40(Web Server issue):W597-603
references.pmid: 22661580
references.citation: J Vis Exp. 2008 Oct 31;(20):
references.pmid: 19078935