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，并且 多种疾病是由KIR通道基因突变引起的。的长期目标是 这个项目是为了了解未被研究的pH敏感Kir4的分子细节。 子家庭频道。以前，我定义了大块阴离子的分子机制 Kir2通道的脂质调节。这些以前的研究是由我的 一种新的真核细胞Kir2表达系统及纯化方案的建立 渠道，以及独特地结合了(1)计算的多种方法 对接模拟，(2)重组蛋白的功能分析 合成脂质体；(3)高分辨结构测定。这些研究 为获得对未充分研究的pH敏感Kir4的新理解提供模板 分子水平上的通道。我们将建立一个高效的表达系统 和用于大规模提纯Kir4.2 Homo-and的纯化方案 未得到充分研究和开发的Kir4.2/Kir5.1杂四聚体蛋白 特定于功能状态的纳米体，不仅将促进结构 在某些功能状态下的测定，但也有治疗潜力。 一旦建立起来，我们将利用同样的“三重组合”的方法来 确定这些未被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.