Structure/function analysis of understudied pH-sensitive Kir4.2 channels in vitro with conformation specific nanobodies

项目摘要 内向整流钾（Kir）通道调节许多组织的兴奋性， Kir通道基因的突变导致多种疾病。的长期目标 该项目旨在了解未充分研究的pH敏感Kir 4的分子细节 子家庭频道以前，我定义了本体阴离子的分子机制， Kir 2通道的脂质调节。这些先前的研究是由我的 Kir 2真核表达系统的建立及纯化方法的研究 通道，以及独特地结合多种方法（1）计算 对接模拟，（2）重组纯化蛋白的功能测定， 合成脂质体，和（3）高分辨率结构测定。这些研究 为获得对未充分研究的pH敏感Kir 4的新理解提供了模板 分子水平上的通道。我们将建立一个高效的表达系统 以及用于大规模纯化Kir4.2同源物和 Kir4.2/Kir5.1异源四聚体蛋白的研究不足， 对功能状态特异的纳米抗体， 在某些功能状态下的决定，但也有治疗潜力。 一旦确立，我们将利用同样的“三重结合”办法， 确定这些未充分研究的通道调节和功能的分子细节。

Project Summary Inward rectifying potassium (Kir) channels regulate excitability in many tissues, and multiple diseases result from mutations of Kir channel genes. The long-term goal of this project is to understand the molecular details of understudied pH-sensitive Kir4 sub-family channels. Previously, I defined molecular mechanisms of bulk anionic lipid regulation of Kir2 channels. These previous studies were made possible by my establishing a new expression system and purification protocols for eukaryotic Kir2 channels, as well as uniquely combining the multiple methods of (1) computational docking simulations, (2) functional assays of purified proteins reconstituted in synthetic liposomes, and (3) high resolution structure determination. These studies provide a template for gaining new understanding of understudied pH-sensitive Kir4 channels at the molecular level. We will establish an efficient expression system and a purification protocol for large scale purifications of Kir4.2 homo- and Kir4.2/Kir5.1 hetero-tetramer proteins that are understudied as well as develop nanobodies specific to functional states that will not only facilitate structure determinations in certain functional states but also have therapeutic potentials. Once established, we will utilize the same `triple combination' of approaches to determine molecular details of these understudied channel regulation and function.