Graduate Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 2012 May 25;287(22):17985-95. doi: 10.1074/jbc.M112.349019. Epub 2012 Mar 27.
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.
K(ATP) 通道（SUR1/Kir6.2）（4）（磺酰脲受体 1/钾内向整流器 6.2）对胰腺β细胞的代谢状态作出反应，调节膜电位和胰岛素分泌。两个亚基的突变通过过度激活孔导致新生儿糖尿病。活性过高的通道由于葡萄糖代谢增加而不能适当关闭；因此，β细胞超极化限制胰岛素释放。K(ATP) 通道被 Kir6.2 孔内的 ATP 结合抑制，并通过 SUR1 的镁核苷酸以不确定的机制被刺激。格列本脲（GBC），一种磺酰脲，被用作构象探针，以比较野生型与 Q1178R 和 R1182Q SUR1 突变体的核苷酸作用。GBC 与无孔受体以高亲和力结合，推测在朝向内部的 ATP 结合盒构象中；MgATP 通过向向外的构象转变降低结合亲和力。为了在平衡条件下确定核苷酸亲和力，在非水解条件下，消除了 Mg(2+)。四态平衡模型描述了变构连接。对于野生型，ATP(4-)的 K(D)分别为1 与 12 mM，对于 Q1178R 为10。连接常数为~10，这意味着外向构象以较低的亲和力结合 GBC，对于 Q1178R 为 9-10 nM。因此，核苷酸不能完全抑制 GBC 结合。据报道，通道开放剂的结合需要 ATP 水解，但 SUR1 选择性激动剂二氮嗪浓度依赖性地增强了 ATP(4-)的作用。八态模型描述了二氮嗪与 ATP(4-)结合之间的联系；二氮嗪显著增加了 Q1178R 对 ATP(4-)的亲和力，并且增加了 ATP(4-)对二氮嗪的结合。与野生型相比，突变体的 NBD2（而不是 NBD1）对 ATP（和 ADP）具有更高的亲和力（有或没有 Mg(2+)）。因此，突变体花费更多的时间处于核苷酸结合构象中，对 GBC 的亲和力降低，从而激活孔。
