Structural basis of KATP channel gating

项目摘要 ATP敏感性钾（KATP）通道将细胞代谢与膜兴奋性偶联，并且对于许多生物学功能至关重要。 生理功能。它们是由四个内向整流的K+形成的独特的膜蛋白复合物 通道（Kir6.1或Kir6.2）亚基和四个磺酰脲受体（SUR 1或SUR 2）亚基。这笔赠款的重点是 KATP通道由Kir6.2和SUR 1组成，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导致先天性高胰岛素血症， 低血糖，而功能获得性突变导致新生儿糖尿病，在严重的情况下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2型糖尿病风险。我们的长期目标是了解结构-功能关系， KATP通道，以便开发针对KATP功能障碍引起的疾病的基于机制的疗法。 在过去的三个供资周期中，我们在实现这一目标方面取得了重大进展。尤其是， 使用单粒子低温电子显微镜（cryoEM），我们最近获得了高分辨率的结构， 通道与生理抑制剂ATP和抗糖尿病药物格列本脲（格列本脲）结合。这 为该领域开辟了新的篇章，使我们能够了解KATP通道组装的结构基础， 在健康和疾病中的门控，在接近原子的细节和在全通道结构的上下文中。我们集团 通过将我们的cryoEM能力与广泛的分子， 生物化学和生物物理学的工具和知识，我们已经积累了在这个过程中的赠款。在这次更新中， 应用程序，我们建议解决最重要的，但具有挑战性的问题仍然在外地。我们 总体假设是，SUR 1通过特异性调节Kir6.2的功能， 由生理学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的方法来测试的假设， 三个相互交织但又独立的具体目标。(1)阐明KATP通道组装机制， 我们的冷冻电镜结构(2)识别和监测SUR 1、Kir6.2和配体之间的相互作用，这些相互作用对于 渠道打开和关闭，以了解管理渠道门控的结构机制。(3)确定 通过cryoEM分析KATP通道的开放状态结构，以了解门控期间的构象转变。的 该提案有一个强有力的科学前提，建立在我们严格的初步和已发表的研究以及 仔细阅读文献。该提案是创新的，因为它将产生新的结构和概念测试 关于通道门控和组装的新机制假说源自最近的cryoEM结构。 成功的结果将对推进我们对渠道监管的结构性知识产生重大影响， 促进针对由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ABC转运蛋白和对人类健康重要的离子通道的广泛影响。

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.