Structural basis of KATP channel gating

KATP通道门控的结构基础

• 批准号: 9914800
• 负责人: Show-Ling Shyng
• 金额: $ 43.53万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-07 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9914800
• 关键词: ATP-Binding Cassette Transporters; Affect; Antidiabetic Drugs; Binding; Biochemical; Biogenesis; Blood Vessels; Brain; CRSP3 gene; Carbamazepine; Cells; Cellular Metabolic Process; Chemicals; Complex; Cryoelectron Microscopy; Data; Defect; Detergents; Developmental Delay Disorders; Disease; Distal; Drug Design; Epilepsy; Functional disorder; Funding; Genes; Genetic Polymorphism; Glucose; Glyburide; Goals; Grant; Health; Heart; Hormone secretion; Human; Hypoglycemia; Impairment; Ion Channel; Knowledge; Lead; Ligands; Mediating; Membrane; Membrane Proteins; Methods; MgADP; MgATP; Molecular; Molecular Chaperones; Monitor; Mutation; N-terminal; Non-Insulin-Dependent Diabetes Mellitus; Nucleotides; Outcome; Persistent Hyperinsulinemia Hypoglycemia of Infancy; Pharmaceutical Preparations; Pharmacology; Phosphatidylinositol 4,5-Diphosphate; Physiological; Positioning Attribute; Potassium; Potassium Channel; Publishing; Regulation; Resolution; Review Literature; Risk; Structure; Structure of beta Cell of islet; Structure-Activity Relationship; Surface; Syndrome; Testing; Therapeutic; Transmembrane Domain; base; biochemical tools; biophysical tools; conformational conversion; crosslink; design; dimer; gain of function mutation; glucose metabolism; hormone regulation; human disease; improved; inhibitor/antagonist; innovation; insulin secretion; inward rectifier potassium channel; ischemic injury; loss of function mutation; molecular dynamics; mutant; nanodisk; neonatal diabetes mellitus; novel; particle; preservation; protein complex; reconstitution; repaglinide; sulfonylurea receptor; trafficking

PROJECT SUMMARY ATP-sensitive potassium (KATP) channels couple cell metabolism to membrane excitability and are critical for many physiological functions. They are unique membrane protein complexes formed by four inwardly rectifying K+ channel (Kir6.1 or Kir6.2) subunits and four sulfonylurea receptor (SUR1 or SUR2) subunits. This grant is focused on KATP channels consisting of Kir6.2 and SUR1, which have a key role in glucose-stimulated insulin secretion in pancreatic β-cells. Loss-of-function mutations in these channels cause congenital hyperinsulinism and hypoglycemia, whereas gain-of-function mutations cause neonatal diabetes and in severe cases DEND (Developmental delay, Epilepsy, and Neonatal Diabetes) syndrome. In addition, KATP gene polymorphisms increase risk for type 2 diabetes. Our long-term goal is to understand the structure-function relationship of KATP channels in order to develop mechanism-based therapies for disease caused by KATP dysfunction. Over the past three funding cycles, we have made significant progress towards this goal. Most particularly, using single-particle cryo-electron microscopy (cryoEM) we recently obtained high-resolution structures of the channel bound with the physiological inhibitor ATP and the anti-diabetic drug glibenclamide (glyburide). This opened a new chapter for the field, enabling us to understand the structural basis of KATP channel assembly and gating in health and disease, at near atomic detail and in the context of full channel structure. Our group is uniquely positioned to help lead this effort by integrating our cryoEM capability with the extensive molecular, biochemical, and biophysical tools and knowledge we have amassed over the course of this grant. In this renewal application, we propose to tackle the most important yet challenging problems remaining in the field. Our overarching hypothesis is that SUR1 assembles with and regulates the function of Kir6.2 through specific structural interactions that are regulated by physiological and pharmacological ligands. We will use a combination of structural, molecular dynamics simulation and functional approaches to test the hypothesis in three interwoven but independent Specific Aims. (1) Elucidate KATP channel assembly mechanisms guided by our cryoEM structures. (2) Identify and monitor interactions between SUR1, Kir6.2, and ligands that are critical for channel opening and closure to understand the structural mechanisms governing channel gating. (3) Determine open state structures of KATP channels by cryoEM to understand the conformational transition during gating. The proposal has a strong scientific premise built on our rigorous preliminary and published studies as well as a careful review of the literature. The proposal is innovative as it will generate new structures and test conceptually novel mechanistic hypotheses on channel gating and assembly emanated from the recent cryoEM structures. Successful outcome will have significant impact on advancing our structural knowledge of channel regulation to facilitate mechanistic drug design for disease caused by KATP channel dysfunction. Further, the outcome will have broad implications for many other ABC transporters and ion channels important for human health.

项目摘要 ATP敏感钾（KATP）通道情侣细胞代谢与膜令人兴奋，对许多人至关重要 身体功能。它们是独特的膜蛋白复合物，由四个内部整流K+形成 通道（Kir6.1或Kir6.2）亚基和四个磺酰脲受体（SUR1或SUR2）亚基。这笔赠款是集中的 在由KIR6.2和SUR1组成的KATP通道上，它们在葡萄糖刺激的胰岛素分泌中具有关键作用 胰腺β细胞。这些渠道的功能丧失突变会导致先天性高胰岛素和 低血糖，而功能收益突变会导致新生儿糖尿病，在严重的情况下导致 （发育延迟，癫痫和新生儿糖尿病）综合征。另外，KATP基因多态性 增加2型糖尿病的风险。我们的长期目标是了解结构功能的关系 KATP通道以开发基于机制的KATP功能障碍引起的疾病疗法。 在过去的三个资金周期中，我们已向这一目标取得了重大进步。最特别的， 使用单粒子冷冻电子显微镜（冷冻）我们最近获得了高分辨率结构 与物理抑制剂ATP和抗糖尿病药物Glibenclamide（Glyburide）结合的通道。这 为该领域开了一个新篇章，使我们能够了解KATP渠道组件的结构基础和 健康和疾病的门控，几乎原子细节以及完整的渠道结构的背景。我们的小组是 通过将我们的冷冻功能与广泛的分子，众所周知，独特的位置，可以帮助您领导这一努力 我们在这笔赠款过程中积累的生化和生物物理工具和知识。在此续约中 应用，我们建议解决该领域剩下的最重要的挑战问题。我们的 总体假设是SUR1组装并与Kir6.2的功能通过特定 由物理和药物配体调节的结构相互作用。我们将使用一个 结构，分子动力学模拟和功能方法的结合，以检验假设 三个交织但独立的特定目的。 （1）阐明由KATP通道组装机制引导的 我们的冷冻结构。 （2）识别和监视Sur1，Kir6.2和配体之间至关重要的配体之间的相互作用 通道打开和关闭，以了解有关渠道门控的结构机制。 （3）确定 Cryoem通过Cryoem的开放状态结构了解门控过程中的构象过渡。这 提案具有强大的科学前提，基于我们严格的初步和发表的研究以及 仔细审查文献。该提案具有创新性，因为它将生成新的结构并在概念上进行测试 最近的冷冻结构散发出关于通道门控和组装的新机械假设。 成功的结果将对推进我们对渠道调节的结构知识的影响产生重大影响 促进由KATP通道功能障碍引起的疾病的机械药物设计。此外，结果将有 对许多其他ABC转运蛋白和离子渠道对人类健康很重要的含义。