title: Expression and purification of recombinant human inward rectifier K+ (KCNJ) channels in Saccharomyces cerevisiae.
abstract: The inward rectifier family of potassium (KCNJ) channels regulate vital cellular processes including cell volume, electrical excitability, and insulin secretion. Dysfunction of different isoforms have been linked to numerous diseases including Bartter's, Andersen-Tawil, Smith-Magenis Syndromes, Type II diabetes mellitus, and epilepsy, making them important targets for therapeutic intervention. Using a family-based approach, we succeeded in expressing 10 of 11 human KCNJ channels tested in Saccharomyces cerevisiae. GFP-fusion proteins showed that these channels traffic correctly to the plasma-membrane suggesting that the protein is functional. A 2-step purification process can be used to purify the KCNJ channels to >95% purity in a mono-dispersed form. After incorporation into liposomes, (86)Rb(+) flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels.
authors.affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK.
authors.forename: Nazzareno
authors.initials: N
authors.lastname: D'Avanzo
authors.forename: Wayland W L
authors.initials: WW
authors.lastname: Cheng
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Xia
authors.forename: Liang
authors.initials: L
authors.lastname: Dong
authors.forename: Pavel
authors.initials: P
authors.lastname: Savitsky
authors.forename: Colin G
authors.initials: CG
authors.lastname: Nichols
authors.forename: Declan A
authors.initials: DA
authors.lastname: Doyle
mesh_terms: D001671:Biochemistry / Q000379:methods*; D003001:Cloning, Molecular; D006801:Humans; D024661:Potassium Channels, Inwardly Rectifying / Q000302:isolation & purification* / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins