Structure-Function Analysis of the Sulfonylurea Receptor

磺酰脲受体的结构-功能分析

• 批准号: 6551811
• 负责人: WANDA H VILA-CARRILES
• 金额: $ 2.9万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-16 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6551811
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; adenosinetriphosphatase; autoradiography; diabetes mellitus; enzyme activity; glucose metabolism; hydrolysis; hypoglycemic agents; insulin; neuroendocrine system; pancreatic islet function; potassium channel; predoctoral investigator; protein purification; protein structure function; receptor binding; receptor coupling; receptor expression; sulfonylurea; western blottings; yeast two hybrid system; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; adenosinetriphosphatase; autoradiography; diabetes mellitus; enzyme activity; glucose metabolism; hydrolysis; hypoglycemic agents; insulin; neuroendocrine system; pancreatic islet function; potassium channel; predoctoral investigator; protein purification; protein structure function; receptor binding; receptor coupling; receptor expression; sulfonylurea; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): ATP-sensitive potassium channels (KATP channels) in pancreatic beta-cells regulate insulin secretion in response to changes in glucose metabolism. KATP channels are composed of 2 subunits: an inward rectifier (KIR6.2) which forms the pore and a sulfonylurea receptor, SUR1, that regulates channel activity giving sensitivity to MgADP, sulfonylureas (SUs) and potassium channel openers (KCOs). Some forms of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) are caused by mutations in KATP channel subunits that result in loss of channel activity. KATP channels set the membrane potential and loss of activity leads to depolarization that allows voltage-gated Ca2+ channels to open, increasing the Ca2+ levels enough to release of insulin. KATP channels are the targets for SUs used to treat non-insulin dependent diabetes mellitus because they close KATP channels and cause insulin secretion. KCOs inhibit insulin secretion by opening KATP channels and are used to treat PHHI and hyperinsulinemic states. Both of these drugs bind to SUR1, the regulatory subunit, which is an ATP binding cassette protein with two nucleotide binding folds (NBFs) and an ABC signature sequence in each NBF. The long-term objectives of this proposal are to understand the structure and regulation of KATP channels. Studies on the ATP binding and hydrolytic properties of SUR1 are critical for understanding how these channels are regulated by nucleotides, how novel compounds can be developed to regulate cellular electrical activity, and they should provide insight into diabetes and other disorders of glucose metabolism. These studies will also help us understand the physiological role of KATP channels in the neuro-endocrine system. My objective is to perform a structure-function analysis of SUR1, specifically I will pursue the following aims: (1) Determine the domains of SUR1 and K IR6.2 that are in close proximity to 1251-azidoglibenclamide, a derivative of a commonly used hypoglycemic agent that binds tightly to SUR1, covalently photolabeling it and K IR6.2; (2) Interchange the signature sequences of SUR1 to determine if they are required for ATP binding, hydrolysis, and regulation of channel activity; (3) Pursue my early observation that there is an interaction between the N-terminus of K IR6.2 and the C-terminus of SUR1. This will be done using a two-hybrid system approach and GST-"pull-downs"; (4) Attempt to express and purify SUR1 using the yeast S. cerevisiae in order to develop assays to measure its ATPase activity directly.

描述（由申请人提供）：胰腺β细胞中的ATP敏感钾通道（KATP通道）调节胰岛素分泌，以响应葡萄糖代谢的变化。 KATP通道由2个亚基组成：一个内向整流器（KIR6.2），形成孔和磺酰尿素受体SUR1，该孔调节通道活性，从而使MGADP，Sulfonylureas（SUS）（SUS）和钾通道（KCOS）敏感。 婴儿期的某些形式的持续性高胰岛素低血糖（PHHI）是由KATP通道亚基突变引起的，导致通道活性的丧失。 KATP通道设定了膜电位和活性损失导致去极化，从而使电压门控的Ca2+通道打开，增加了足够的Ca2+水平以释放胰岛素。 KATP通道是用于治疗非胰岛素依赖性糖尿病的SUS的靶标，因为它们关闭KATP通道并引起胰岛素分泌。 KCO通过打开KATP通道抑制胰岛素分泌，并用于治疗PHHI和高胰岛素态。 这两种药物都与SUR1结合，SUR1是调节亚基，这是一种ATP结合盒蛋白，具有两个核苷酸结合褶皱（NBF）和每个NBF中的ABC签名序列。 该提案的长期目标是了解KATP渠道的结构和调节。 关于SUR1的ATP结合和水解特性的研究对于理解这些通道如何受核苷酸调节，如何开发新化合物来调节细胞电活动至关重要，并且应提供对糖尿病和葡萄糖代谢的其他疾病的见识。 这些研究还将帮助我们了解KATP通道在神经内分泌系统中的生理作用。 我的目标是对SUR1进行结构功能分析，特别是我将追求以下目的：（1）确定SUR1和K IR6.2的域，这些域与1251-齐adoglibenclenc酰胺紧密接近，这是一种常用的降低性降低性降低药剂的衍生物，与Sur1紧密结合，与SUR1紧密结合，与Sur1紧密结合，共凝聚，klypoptalbalency PhotolabeLabeLabeLabeLecrabelIs和Kir6.2; 2 ir6.2; （2）互换SUR1的签名序列，以确定它们是否需要ATP结合，水解和通道活性调节； （3）追求我的早期观察，即K IR6.2的N端与Sur1的C端之间存在相互作用。 这将使用两杂交系统方法和GST-“下拉”来完成； （4）尝试使用酵母菌酿酒酵母表达和净化SUR1，以开发直接测量其ATPase活性的测定。