Two Neonatal Diabetes Mutations on Transmembrane Helix 15 of SUR1 Increase Affinity for ATP and ADP at Nucleotide Binding Domain 2

Abstract KATP 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. KATP 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, Mg2+ was eliminated. A four-state equilibrium model describes the allosteric linkage. The KD for ATP4− 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 ATP4− action. An eight-state model describes linkage between diazoxide and ATP4− binding; diazoxide markedly increases the affinity of Q1178R for ATP4− and ATP4− augments diazoxide binding. NBD2, but not NBD1, has a higher affinity for ATP (and ADP) in mutant versus wild type (with or without Mg2+). Thus, the mutants spend more time in nucleotide-bound conformations, with reduced affinity for GBC, that activate the pore.