Correlating structure and function in KATP channel isoforms

项目摘要 ATP敏感性钾通道（KATP）由胞内核苷酸ATP和ADP门控， 具有膜兴奋性的能量学，用于管理对能量至关重要的广泛生理过程 体内平衡KATP通道是由四个内向整流钾离子组成的独特的八聚体膜蛋白复合物 通道（Kir6.1或Kir6.2）亚基和四个磺酰脲受体（SUR 1、SUR 2A或SUR 2B）亚基。各种 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/SUR 1、Kir6.2/SUR 2A和Kir6.2/SUR 2B通道。 Kir6.1/SUR 2B组合，代表主要的胰腺、心脏和血管平滑肌亚型， 分别各种KATP通道中的基因突变是许多内分泌、心血管和 神经和肌肉疾病，如Cantú综合征，一种严重的多效性全身性低血压 由血管Kir6.1/SUR 2B KATP通道中的功能获得突变引起的疾病。一个中心目标， KATP通道领域是为了了解KATP通道亚型的结构-功能关系，以便开发 针对KATP通道功能障碍引起的疾病的基于机制的同种型特异性疗法。的一个主要障碍 进展是缺乏高分辨率的沟道结构。最近，我们通过解决 使用cryoEM将胰腺Kir6.2/SUR 1通道的3D结构提高到近原子分辨率。在这个新 应用，我们试图进行这种势头，并确定其他KATP通道亚型的结构。我们 假设比较和对比相关KATP通道复合物将揭示一般设计 原理，使KATP通道作为ATP/ADP传感器和具体的机制， 不同KATP通道亚型的独特门控特性和药理学。我们将使用一个 多管齐下的方法，结合单粒子cryoEM，分子动力学模拟， 化合物筛选和三个独立但整合的特异性目的中的功能测定。(1)确定 载脂蛋白、转运蛋白结合和激活剂中KATP通道亚型的cryoEM结构和构象动力学- 约束状态。(2)测试KATP通道亚型特异性生物物理特性的机制假设， 核苷酸敏感性和药理学。(3)进行基于结构的概念验证药物发现研究 针对血管KATP通道中Cantú突变的靶向药理学。科学的前提是 该提案是建立在丰富的KATP通道文献和严格的初步和发表的研究， 申请人及其合作研究者和合作者。该提案是创新的，因为它旨在产生新的 结构，建立KATP门控调节机制的新概念，并发现新的血管KATP 用于Cantú患者的通道抑制剂。成功的结果将推进KATP调节的结构知识， 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 领域这也将对其他ABC转运蛋白和对人类健康至关重要的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.