Structural basis of KATP channel gating

项目总结 ATP敏感钾(KATP)通道将细胞代谢与膜兴奋性结合在一起，对许多细胞来说是至关重要的 生理功能。它们是由四个内向整流钾形成的独特的膜蛋白复合体 通道(Kir6.1或Kir6.2)亚基和四个磺酰脲受体(SUR1或SUR2)亚基。这笔赠款的重点是 在由Kir6.2和SUR1组成的KATP通道上，KATP通道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和 低血糖，而功能获得突变会导致新生儿糖尿病，在严重情况下会导致糖尿病 (发育迟缓、癫痫和新生儿糖尿病)综合征。此外，KATP基因的多态性 增加患2型糖尿病的风险。我们的长期目标是理解结构与功能的关系 KATP通道，以开发基于机制的治疗KATP功能障碍引起的疾病。 在过去的三个供资周期中，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 翻开了该领域的新篇章，使我们能够了解KATP通道组装和 在健康和疾病中，以接近原子的细节和在全通道结构的背景下进行选通。我们的小组是 通过将我们的低温EM能力与广泛的分子、 生物化学、生物物理工具和我们在这笔赠款过程中积累的知识。在这次更新中 在这方面，我们建议解决该领域仍然存在的最重要但具有挑战性的问题。我们的 最重要的假设是，SUR1通过特定的基因与Kir6.2组装并调节其功能 由生理和药理配体调节的结构相互作用。我们将使用 结合结构、分子动力学模拟和功能方法来检验这一假说 三个相互交织但又相互独立的具体目标。(1)阐明KATP通道组装机制 我们的低温电子显微镜结构。(2)识别和监控SUR1、Kir6.2和配体之间的相互作用，这些相互作用对 渠道开启和关闭，以了解支配渠道门控的结构机制。(三)确定 用低温电子显微镜研究KATP通道的开放态结构，以了解门控过程中的构象转变。这个 提案有很强的科学前提，建立在我们严谨的初步和已发表的研究以及 仔细阅读相关文献。该提议是创新的，因为它将产生新的结构并在概念上进行测试 最近的低温电子显微镜结构产生了关于沟道门控和组装的新的机械假说。 成功的结果将对我们推进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械性药物设计。更进一步，结果将会是 对许多其他ABC转运蛋白和离子通道的广泛影响对人类健康很重要。

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.