Correlating structure and function in KATP channel isoforms

项目总结 由细胞内核苷酸ATP和ADP调控的ATP敏感钾(KATP)通道，偶联细胞 具有膜兴奋性的能量学，用于控制对能量至关重要的广泛生理过程 动态平衡。KATP通道是由四种内向整流钾离子组成的独特的异构膜蛋白复合体 通道(Kir6.1或Kir6.2)亚基和四个磺酰脲受体(SUR1、SUR2A或SUR2B)亚基。五花八门 Kir6.x/SURx组合产生具有不同组织分布、核苷酸敏感性、 和药理学。最突出的KATP通道是Kir6.2/SUR1、Kir6.2/SUR2A和 Kir6.1/SUR2B组合，代表主要的胰腺、心脏和血管平滑肌亚型， 分别进行了分析。不同KATP通道中的基因突变是许多内分泌、心血管和 神经元和肌肉疾病，如Cantú综合征，一种严重的多效性全身性低血压 血管Kir6.1/SUR2B KATP通道功能突变导致的疾病。一个核心目标是 KATP通道场是为了了解KATP通道异构体的结构-功能关系才能发展起来的 KATP通道功能障碍引起的疾病的基于机制的、异构体特异性的治疗。一个关键障碍是 进展是缺乏高分辨率的通道结构。最近，我们已经打破了这一障碍，解决了 胰腺Kir6.2/SUR1通道的三维结构到近原子分辨率的低温电子显微镜。在这个新的 应用，我们寻求携带这一势头，并确定其他KATP通道异构体的结构。我们 假设比较和对比相关的KATP通道复合体将揭示总体设计 允许KATP通道作为ATP/ADP传感器运行的原理和基础的特定机制 不同KATP通道亚型独特的门控特性和药理作用。我们将使用一个 多管齐下，结合单粒子低温电子显微镜、分子动力学模拟，在硅胶中 化合物筛选，以及三个独立但综合的特定目标的功能分析。(一)确定 载脂蛋白、抑制剂结合和激活剂中KATP通道亚型的低温EM结构和构象动力学 束缚态。(2)验证KATP通道异构体特异性生物物理性质的机制假说， 核苷酸敏感性和药理学。(3)进行基于概念验证结构的药物发现研究 针对血管KATP通道中Cantú突变的靶向药理学。科学的前提是 该提案建立在丰富的KATP渠道文献和来自 申请人和她的合作调查员和合作者。这项提议是创新的，因为它旨在产生新的 结构，建立KATP门控调控机制的新概念，发现新的血管KATP 用于Cantú患者的通道抑制剂。成功的结果将促进对KATP调控的结构性知识 健康和疾病，并将KATP药理学转向基于结构的范式，以显著影响 菲尔德。这也将对其他对人类健康至关重要的ABC转运蛋白和离子通道产生广泛的影响。

PROJECT SUMMARY ATP-sensitive potassium (KATP) channels, gated by intracellular nucleotides ATP and ADP, couple cell energetics with membrane excitability to govern a wide range of physiological processes vital to energy homeostasis. KATP channels are unique hetero-octameric membrane protein complexes of four inward rectifier K+ channel (Kir6.1 or Kir6.2) subunits and four sulfonylurea receptor (SUR1, SUR2A, or SUR2B) subunits. Various Kir6.x/SURx combinations generate KATP channel isoforms with distinct tissue distribution, nucleotide sensitivity, and pharmacology. The most prominent KATP channels are those of Kir6.2/SUR1, Kir6.2/SUR2A, and Kir6.1/SUR2B combinations, representing the major pancreatic, cardiac, and vascular smooth muscle isoforms, respectively. Genetic mutations in the various KATP channels underlie a number of endocrine, cardiovascular, and neuronal, and muscular diseases, as exemplified by Cantú syndrome, a severe pleiotropic systemic hypotension disorder caused by gain of function mutations in the vascular Kir6.1/SUR2B KATP channel. A central goal in the KATP channel field is to understand the structure-function relationship of KATP channel isoforms in order to develop mechanism-based, isoform-specific therapies for disease caused by KATP channel dysfunction. A key barrier to progress has been a lack of high resolution channel structures. Recently, we have broken this barrier by resolving 3D structures of the pancreatic Kir6.2/SUR1 channel to near atomic resolutions using cryoEM. In this new application, we seek to carry this momentum and determine structures of the other KATP channel isoforms. We hypothesize that comparing and contrasting related KATP channel complexes will reveal the general design principles that allow KATP channels to operate as ATP/ADP sensors and the specific mechanisms that underlie the unique gating properties and pharmacology of different KATP channel isoforms. We will test the hypothesis using a multipronged approach that combines single-particle cryoEM, molecular dynamics simulations, in silico compound screening, and functional assays in three independent but integrated Specific Aims. (1) Determine cryoEM structures and conformational dynamics of KATP channel isoforms in apo, inhibitors-bound and activators- bound states. (2) Test mechanistic hypotheses on KATP channel isoform-specific biophysical properties, nucleotides sensitivities, and pharmacology. (3) Conduct proof-of-concept structure-based drug discovery studies towards targeted pharmacology for Cantú mutations in vascular KATP channels. The scientific premise of the proposal is built on a wealth of KATP channel literature and rigorous preliminary and published studies from the applicant and her co-investigator and collaborators. The proposal is innovative as it aims to generate new structures, establish new concepts in KATP gating regulation mechanisms, and discover new vascular KATP channel inhibitors for Cantú patients. Successful outcome will advance structural knowledge of KATP regulation in health and disease, and shift KATP pharmacology towards a structure-based paradigm to significantly impact the field. It will also have broad implications for other ABC transporters and ion channels critical for human health.