title: Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer.
abstract: K(ATP) channels link cell metabolism to excitability in many cells. They are formed as tetramers of Kir6.2 subunits, each associated with a SUR1 subunit. We used mutant GFP-based FRET to assess domain organization in channel complexes. Full-length Kir6.2 subunits were linked to YFP or cyan fluorescent protein (CFP) at N or C termini, and all such constructs, including double-tagged YFP-Kir6.2-CFP (Y6.2C), formed functional K(ATP) channels. In intact COSm6 cells, background emission of YFP excited by 430-nm light was ∼6%, but the Y6.2C construct expressed alone exhibited an apparent FRET efficiency of ∼25%, confirmed by trypsin digestion, with or without SUR1 co-expression. Similar FRET efficiency was detected in mixtures of CFP- and YFP-tagged full-length Kir6.2 subunits and transmembrane domain only constructs, when tagged at the C termini but not at the N termini. The FRET-reported Kir6.2 tetramer domain organization was qualitatively consistent with Kir channel crystal structures: C termini and M2 domains are centrally located relative to N termini and M1 domains, respectively. Additional FRET analyses were performed on cells in which tagged full-length Kir6.2 and tagged SUR1 constructs were co-expressed. These analyses further revealed that 1) NBD1 of SUR1 is closer to the C terminus of Kir6.2 than to the N terminus; 2) the Kir6.2 cytoplasmic domain is not essential for complexation with SUR1; and 3) the N-terminal half of SUR1 can complex with itself in the absence of either the C-terminal half or Kir6.2.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Shizhen
authors.initials: S
authors.lastname: Wang
authors.forename: Elena N
authors.initials: EN
authors.lastname: Makhina
authors.forename: Ricard
authors.initials: R
authors.lastname: Masia
authors.forename: Krzysztof L
authors.initials: KL
authors.lastname: Hyrc
authors.forename: Mary Lynn
authors.initials: ML
authors.lastname: Formanack
authors.forename: Colin G
authors.initials: CG
authors.lastname: Nichols
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer; D054086:KATP Channels / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D064233:Sulfonylurea Receptors
chemical_list: D018528:ATP-Binding Cassette Transporters; C577508:Abcc8 protein, mouse; D054086:KATP Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D064233:Sulfonylurea Receptors; C092520:uK-ATP-1 potassium channel