Structural basis of KATP channel gating

KATP通道门控的结构基础

• 批准号: 10339382
• 负责人: Show-Ling Shyng
• 金额: $ 43.83万
• 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/10339382
• 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; 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.

项目概要 ATP 敏感钾 (KATP) 通道将细胞代谢与膜兴奋性耦合起来，对许多细胞至关重要 生理功能。它们是由四个内向整流的 K+ 形成的独特膜蛋白复合物 通道（Kir6.1 或 Kir6.2）亚基和四个磺酰脲受体（SUR1 或 SUR2）亚基。本次补助重点 由 Kir6.2 和 SUR1 组成的 KATP 通道，其在葡萄糖刺激的胰岛素分泌中起关键作用 胰腺β细胞。这些通道的功能丧失突变会导致先天性高胰岛素血症 低血糖，而功能获得性突变会导致新生儿糖尿病，严重时会导致 DEND （发育迟缓、癫痫和新生儿糖尿病）综合征。此外，KATP基因多态性 增加患 2 型糖尿病的风险。我们的长期目标是了解结构与功能的关系 KATP 通道，旨在开发针对 KATP 功能障碍引起的疾病的基于机制的疗法。 在过去的三个融资周期中，我们在实现这一目标方面取得了重大进展。最特别的是， 使用单粒子冷冻电子显微镜（cryoEM），我们最近获得了高分辨率的结构 与生理抑制剂 ATP 和抗糖尿病药物格列本脲（格列本脲）结合的通道。这 为该领域翻开了新的篇章，使我们能够了解KATP通道组装的结构基础并 在接近原子细节和完整通道结构的背景下对健康和疾病进行门控。我们组是 通过将我们的冷冻电镜能力与广泛的分子、 我们在这笔资助过程中积累了生物化学和生物物理工具和知识。在这次更新中 应用程序中，我们建议解决该领域仍然存在的最重要但最具挑战性的问题。我们的 总体假设是 SUR1 通过特定的途径与 Kir6.2 组装并调节其功能 受生理和药理学配体调节的结构相互作用。我们将使用一个 结合结构、分子动力学模拟和功能方法来检验假设 三个相互交织但独立的具体目标。 (1) 阐明KATP通道组装机制 我们的冷冻电镜结构。 (2) 识别并监测 SUR1、Kir6.2 和配体之间的相互作用，这些相互作用对 通道打开和关闭，以了解控制通道门控的结构机制。 (3)确定 通过冷冻电镜打开 KATP 通道的状态结构，以了解门控过程中的构象转变。这 该提案具有强有力的科学前提，建立在我们严格的初步和已发表的研究以及 仔细查阅文献。该提案具有创新性，因为它将产生新的结构并在概念上进行测试 关于通道门控和组装的新机制假设源于最近的冷冻电镜结构。 成功的结果将对提高我们对渠道监管的结构性知识产生重大影响 促进针对 KATP 通道功能障碍引起的疾病的机制药物设计。此外，结果将有 对许多其他对人类健康重要的 ABC 转运蛋白和离子通道具有广泛的影响。