Gating mechanism of a potassium channel (KirBac3.1) studied using high resolution cryo electron crystallography

几乎人体的每一个过程都在某种程度上受到电信号的控制，从我们的心跳方式，肌肉运动方式，到我们的思维方式。这些电信号是由离子通道产生和控制的，离子通道充当电子“纳米开关”，控制钾（K+）和钠（Na+）等带电离子进出细胞的选择性运动。由于这一基本重要性，大量的遗传疾病或“通道病”是离子通道功能缺陷的结果。这些疾病包括囊性纤维化、糖尿病、癫痫和许多其他罕见的心脏、大脑、神经和肾脏疾病。离子通道也有相当大的潜力被开发成纳米级电子器件中的分子开关，因此了解它们是如何打开和关闭的，以及如何控制这个过程是相当有趣的。内整流钾（Kir）通道几乎在身体的每个细胞中表达，并且是这些细胞中发现的基本电活动的基础。这些人体通道的结构研究是极其困难的。然而，相关的“KirBac”通道存在于细菌中，这些通道更适合生化和结构研究。在我们的初步研究中，我们表达和纯化了KirBac3.1，并证明我们能够使用强大的电子显微镜获得该通道在打开和关闭状态下的图像。在这个提议中，我们的目标是使用一种新的最先进的显微镜和KirBac3.1突变蛋白来提高我们能够实现的图像的分辨率。这将使我们能够获得关闭和打开状态下的KirBac3.1通道的高分辨率图像，并解决通道在打开和关闭状态之间改变形状时发生的结构变化。提高我们对这一过程在分子水平上如何发生的理解，将为了解相关K+通道如何打开和关闭以及这一过程如何在疾病状态下变得有缺陷提供重要见解。

Almost every single process in the human body is controlled at some level by electrical signals, from the way our hearts beat, the way our muscles move, to the way we think. These electrical signals are generated and controlled by ion channels which act as electrical 'nano-switches' to control the selective movement of charged ions like potassium (K+) and sodium (Na+) into and out of the cell. As a consequence of this fundamental importance, a large number of genetically inherited diseases or 'channelopathies' are the result of defective ion channel function. These diseases include cystic fibrosis, diabetes, epilepsy and many other rarer diseases of the heart, brain, nerves and kidneys. Ion channels also have considerable potential to be exploited as molecular switches in nanoscale electrical devices and so understanding how they open and close, and how this process is controlled is of considerable interest. Inwardly-rectifying potassium (Kir) channels are expressed in almost every cell of the body and underlie the basic electrical activity which is found in these cells. Structural studies of these human channels is extremely difficult. However, related 'KirBac' channels exist in bacteria and these are much more amenable to biochemical and structural studies. In our preliminary investigations we have expressed and purified KirBac3.1 and shown we are able to obtain images of this channel in both the open and the closed state using a powerful electron microscope. In this proposal we aim to use a new state-of-the-art microscope and mutant KirBac3.1 proteins to improve the resolution of our images we are able to achieve. This will allow us to obtain high-resolution images of the KirBac3.1 channel in both the closed and open states and to address the structural changes which occur as the channel changes shape between the open and closed states. Improving our understanding of how this process occurs at the molecular level will provide a major insight into how related K+ channels open and close and how this process becomes defective in the disease state.