title: Proximal C-terminal domain of sulphonylurea receptor 2A interacts with pore-forming Kir6 subunits in KATP channels.
abstract: Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein-SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254-1545), rSUR2A-CTB (residues 1254-1403) and rSUR2A-CTC (residues 1294-1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358-1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294-1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294-1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 9HN, UK.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Rainbow
authors.forename: Marian
authors.initials: M
authors.lastname: James
authors.forename: Diane
authors.initials: D
authors.lastname: Hudman
authors.forename: Mohammed
authors.initials: M
authors.lastname: Al Johi
authors.forename: Harprit
authors.initials: H
authors.lastname: Singh
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Watson
authors.forename: Ian
authors.initials: I
authors.lastname: Ashmole
authors.forename: Noel W
authors.initials: NW
authors.lastname: Davies
authors.forename: David
authors.initials: D
authors.lastname: Lodwick
authors.forename: Robert I
authors.initials: RI
authors.lastname: Norman
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D005905:Glyburide / Q000494:pharmacology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017136:Ion Transport / Q000187:drug effects; D007668:Kidney; D046911:Macromolecular Substances; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051381:Rats; D011955:Receptors, Drug / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D064233:Sulfonylurea Receptors; D014162:Transfection
chemical_list: C577510:ABCC9 protein, human; D018528:ATP-Binding Cassette Transporters; C577512:Abcc9 protein, rat; D046911:Macromolecular Substances; D010446:Peptide Fragments; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D064233:Sulfonylurea Receptors; D000255:Adenosine Triphosphate; D011188:Potassium; D005905:Glyburide