title: Reconstitution of KCNE1 into lipid bilayers: comparing the structural, dynamic, and activity differences in micelle and vesicle environments.
abstract: KCNE1 (minK), found in the human heart and cochlea, is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study, a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (POPG) was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (K(V)7.1) voltage-gated potassium channel led to nativelike modulation of the channel. Circular dichroism spectroscopy demonstrated that the percent helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles than for the previously described structure in 1.0% 1-myristoyl-2-hydroxy-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (LMPG) micelles. SDSL electron paramagnetic resonance spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu59, and Ser64 of KCNE1 in both POPC/POPG vesicles and LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water inaccessible in vesicles but found to be water accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers but also demonstrate the need to refine this structure using data derived from the bilayer-reconstituted protein to more accurately define its native structure. This work establishes the basis for such future studies.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Coey
authors.forename: Indra D
authors.initials: ID
authors.lastname: Sahu
authors.forename: Thusitha S
authors.initials: TS
authors.lastname: Gunasekera
authors.forename: Kaylee R
authors.initials: KR
authors.lastname: Troxel
authors.forename: Jaclyn M
authors.initials: JM
authors.lastname: Hawn
authors.forename: Max S
authors.initials: MS
authors.lastname: Swartz
authors.forename: Marilyn R
authors.initials: MR
authors.lastname: Wickenheiser
authors.forename: Ro-jay
authors.initials: RJ
authors.lastname: Reid
authors.forename: Richard C
authors.initials: RC
authors.lastname: Welch
authors.forename: Carlos G
authors.initials: CG
authors.lastname: Vanoye
authors.forename: Congbao
authors.initials: C
authors.lastname: Kang
authors.forename: Charles R
authors.initials: CR
authors.lastname: Sanders
authors.forename: Gary A
authors.initials: GA
authors.lastname: Lorigan
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008823:Micelles; D008845:Microinjections; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: C489622:KCNE1 protein, human; D008051:Lipid Bilayers; D008081:Liposomes; D008823:Micelles; D050505:Mutant Proteins; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; C552372:lysomyristoylphosphatidylglycerol; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
references.citation: J Gen Physiol. 2006 Dec;128(6):721-9
references.pmid: 17130521
references.citation: Biochem J. 1997 Jul 15;325 ( Pt 2):475-9
references.pmid: 9230130
references.citation: Neuropharmacology. 2004 Nov;47(6):787-821
references.pmid: 15527815
references.citation: Biochim Biophys Acta. 2010 Dec;1797(12):1894-900
references.pmid: 20959113
references.citation: Biophys J. 2003 May;84(5):3190-6
references.pmid: 12719248
references.citation: Biochemistry. 1981 Jan 6;20(1):33-7
references.pmid: 7470476
references.citation: J Physiol. 2006 Feb 1;570(Pt 3):455-67
references.pmid: 16308347
references.citation: Nat Genet. 1997 Nov;17(3):338-40
references.pmid: 9354802
references.citation: Physiology (Bethesda). 2005 Dec;20:408-16
references.pmid: 16287990
references.citation: Biochemistry. 2008 Aug 5;47(31):7999-8006
references.pmid: 18611041
references.citation: Neuron. 2004 Jun 24;42(6):927-37
references.pmid: 15207237
references.citation: Biopolymers. 2008 May;89(5):392-400
references.pmid: 17896349
references.citation: Biochemistry. 2003 Feb 11;42(5):1254-65
references.pmid: 12564928
references.citation: Biochemistry. 2007 Oct 16;46(41):11459-72
references.pmid: 17892302
references.citation: Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1667-71
references.pmid: 8127863
references.citation: Science. 2003 Jan 10;299(5604):251-4
references.pmid: 12522251
references.citation: J Biol Chem. 2003 Apr 4;278(14):11739-45
references.pmid: 12529362
references.citation: Biochemistry. 2002 Oct 1;41(39):11543-51
references.pmid: 12269798
references.citation: Pediatr Nephrol. 2004 Nov;19(11):1194-201
references.pmid: 15365806
references.citation: J Gen Physiol. 2000 Sep;116(3):379-90
references.pmid: 10962015
references.citation: J Biol Chem. 2002 Jul 12;277(28):25187-94
references.pmid: 11994278
references.citation: J Gen Physiol. 2007 Feb;129(2):121-33
references.pmid: 17227916
references.citation: Nature. 1996 Nov 7;384(6604):80-3
references.pmid: 8900283
references.citation: Anal Biochem. 1990 Nov 15;191(1):110-8
references.pmid: 2077933
references.citation: Biochemistry. 1987 Apr 21;26(8):2117-30
references.pmid: 3040081
references.citation: J Gen Physiol. 2010 Jun;135(6):607-18
references.pmid: 20479109
references.citation: Biochemistry. 1999 Oct 12;38(41):13707-16
references.pmid: 10521278
references.citation: Biophys J. 1997 Nov;73(5):2588-94
references.pmid: 9370453
references.citation: J Biol Chem. 2002 Apr 19;277(16):14068-76
references.pmid: 11825894
references.citation: Biophys J. 1998 Feb;74(2 Pt 1):918-30
references.pmid: 9533703
references.citation: Biochemistry. 1998 Jun 2;37(22):8121-31
references.pmid: 9609707
references.citation: Biochemistry. 2002 May 21;41(20):6282-92
references.pmid: 12009889
references.citation: Biochemistry. 1997 Oct 21;36(42):13027-33
references.pmid: 9335564
references.citation: Proc Natl Acad Sci U S A. 2006 May 9;103(19):7292-7
references.pmid: 16648251
references.citation: Nature. 1996 Nov 7;384(6604):78-80
references.pmid: 8900282
references.citation: FASEB J. 2002 Mar;16(3):390-400
references.pmid: 11874988