Challenging the dominant model for ATP regulation of KATP channels

描述（由申请人提供）：该项目的长期目标是了解腺嘌呤核苷酸对atp敏感的K+， KATP通道的调节；短期目标是挑战现行的监管模式。胰腺ß-细胞中腺嘌呤核苷酸、ATP和ADP的水平随葡萄糖代谢速率的变化而变化。KATP通道响应这些变化，是血糖正常控制胰岛素分泌的关键因素。这些通道是用于治疗2型糖尿病的降糖药磺脲类药物的靶点。ABCC8/SUR1或KCNJ11/Kir6.2通道组分的突变是新生儿糖尿病（ND）和新生儿高胰岛素血症（HI）的原因，而这两个亚基的多态性会增加2型糖尿病的风险。用于解释突变和多态性如何改变通道活性的主流调控假设是，SUR1上的ATP水解需要抵消或拮抗ATP对Kir6.2孔的抑制作用。因此，过度活跃的SUR1通过“过度激活”Kir6.2孔产生ND。SUR1 atp酶活性的改变被认为是ABCC8多态性引起的过度激活和风险增加的基础。我们实验室最近的一项研究挑战了主流模型（Ortiz et al， JBC, 2012）。本研究使用两个ND突变体SUR1，没有相关的Kir6.2，来定义ATP和磺酰脲结合与SUR1构象变化之间的变构关系。我们确定ATP水解不是将SUR1转换为刺激构象所必需的，并提出对ATP的亲和力增加是这种疾病的潜在原因。这项研究现在已经扩展到SUR1对ATP的亲和力与临床表型之间存在直接关系；ATP亲和力高于正常水平的SURs与新生儿糖尿病相关，亲和力较低的SURs与先天性高胰岛素血症相关。ATP和磺脲类药物与SUR1相互作用之间的负变抗关系表明，ND患者需要更高剂量的磺脲类药物来实现代谢控制。拟议的工作将通过药理学和电生理学研究将分析扩展到全通道，研究额外的ND SUR1突变体、多态性和具有抑制atp酶活性的取代的SUR1。先前关于ATP类似物对KATP通道功能作用的研究用于支持主流模型，因此提出了额外的工作来确定它们对SUR1构象开关的作用。该项目的总体目标是开发修改现行模型所需的数据，使其能够充分解释正常生理条件下KATP通道的调节，并对理解突变，特别是ABCC8突变如何影响通道功能具有预测价值。

DESCRIPTION (provided by applicant): The long-term objective of this program is to understand the regulation of ATP-sensitive K+, KATP, channels by adenine nucleotides; the short-term goal is to challenge the prevailing regulatory model. The levels of adenine nucleotides, ATP and ADP, in pancreatic ß-cells vary with the rate of glucose metabolism. KATP channels respond to these variations and are key players in the normal control of insulin secretion by blood glucose. These channels are the targets for sulfonylureas, hypoglycemic drugs used to treat type 2 diabetes. Mutations in the ABCC8/SUR1 or KCNJ11/Kir6.2 channel components are causes of neonatal diabetes (ND) and neonatal hyperinsulinism (HI) while polymorphisms in both subunits confer increased risk for type 2 diabetes. The prevailing regulatory hypothesis, used to interpret how mutations and polymorphisms alter channel activity, is that ATP hydrolysis at SUR1 is required to counteract or antagonize the inhibitory action of ATP on the Kir6.2 pore. Thus an overactive SUR1 produces ND by 'hyperactivating' the Kir6.2 pore. Altered SUR1 ATPase activity is proposed to underlie hyperactivation and the increased risk posed by ABCC8 polymorphisms. A recent study from our laboratory challenges the prevailing model (Ortiz et al, JBC, 2012). This study used two ND mutant SURs, without an associated Kir6.2, to define the allosteric relations between ATP and sulfonylurea binding with changes in SUR1 conformation. We established that ATP hydrolysis is not essential to switch SUR1 into a stimulatory conformation and proposed that an increased affinity for ATP is the underlying cause of the disorder. This study has now been extended to show there is a direct relation between the affinity of SUR1 for ATP and clinical phenotype; SURs with greater than normal affinity for ATP correlate with neonatal diabetes, those with lower affinity correlate with congenital hyperinsulinism. The negative allosteric relation between ATP and sulfonylurea interactions with SUR1 underlies the known need for higher doses of sulfonylureas to achieve metabolic control in ND patients. The proposed work will extend the analysis to full channels using pharmacologic and electrophysiologic studies on additional ND SUR1 mutants, polymorphisms, and on SURs with substitutions that inhibit ATPase activity. Previous studies on the action of ATP analogs on KATP channel function are used to support the prevailing model, thus additional work is proposed to define their action on conformational switching of SUR1. The overall objective of the project is to develop the data required to modify the prevailing model so that it can adequately explain regulation of KATP channels under normal physiologic conditions and has predictive value for understanding how mutations, particularly ABCC8 mutations, affect channel function.