title: Two neonatal diabetes mutations on transmembrane helix 15 of SUR1 increase affinity for ATP and ADP at nucleotide binding domain 2.
abstract: K(ATP) channels, (SUR1/Kir6.2)(4) (sulfonylurea receptor type 1/potassium inward rectifier type 6.2) respond to the metabolic state of pancreatic β-cells, modulating membrane potential and insulin exocytosis. Mutations in both subunits cause neonatal diabetes by overactivating the pore. Hyperactive channels fail to close appropriately with increased glucose metabolism; thus, β-cell hyperpolarization limits insulin release. K(ATP) channels are inhibited by ATP binding to the Kir6.2 pore and stimulated, via an uncertain mechanism, by magnesium nucleotides at SUR1. Glibenclamide (GBC), a sulfonylurea, was used as a conformational probe to compare nucleotide action on wild type versus Q1178R and R1182Q SUR1 mutants. GBC binds with high affinity to aporeceptors, presumably in the inward facing ATP-binding cassette configuration; MgATP reduces binding affinity via a shift to the outward facing conformation. To determine nucleotide affinities under equilibrium, non-hydrolytic conditions, Mg(2+) was eliminated. A four-state equilibrium model describes the allosteric linkage. The K(D) for ATP(4-) is ~1 versus 12 mM, Q1178R versus wild type, respectively. The linkage constant is ~10, implying that outward facing conformations bind GBC with a lower affinity, 9-10 nM for Q1178R. Thus, nucleotides cannot completely inhibit GBC binding. Binding of channel openers is reported to require ATP hydrolysis, but diazoxide, a SUR1-selective agonist, concentration-dependently augments ATP(4-) action. An eight-state model describes linkage between diazoxide and ATP(4-) binding; diazoxide markedly increases the affinity of Q1178R for ATP(4-) and ATP(4-) augments diazoxide binding. NBD2, but not NBD1, has a higher affinity for ATP (and ADP) in mutant versus wild type (with or without Mg(2+)). Thus, the mutants spend more time in nucleotide-bound conformations, with reduced affinity for GBC, that activate the pore.
authors.affiliation: Graduate Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: David
authors.initials: D
authors.lastname: Ortiz
authors.forename: Peter
authors.initials: P
authors.lastname: Voyvodic
authors.forename: Lindsay
authors.initials: L
authors.lastname: Gossack
authors.forename: Ulrich
authors.initials: U
authors.lastname: Quast
authors.forename: Joseph
authors.initials: J
authors.lastname: Bryan
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D001665:Binding Sites; D003920:Diabetes Mellitus / Q000235:genetics*; D006801:Humans; D007231:Infant, Newborn; D008958:Models, Molecular; D009154:Mutation; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000235:genetics*; D011955:Receptors, Drug / Q000737:chemistry / Q000235:genetics*; D064233:Sulfonylurea Receptors
chemical_list: C577507:ABCC8 protein, human; D018528:ATP-Binding Cassette Transporters; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate