3-D DESIGN AND ACTION OF CELL MEMBRANE CHANNELS

这项研究的主要目的是更好地理解设计 离子通道及其调节机制的原理 离子流过细胞膜。使用的技术将包括 结构分析的低温电子显微镜和结晶学方法 我们将进一步开发它以获得更高分辨率的信息。我们 他们还试图生长离子通道的三维晶体 适用于X射线衍射研究。来自这些结构的发现 方法将提供一个三维框架，用于将 通过定点突变实验获得的大量数据 结合生理机能研究。 两个特征最好的渠道将作为模型进行研究 系统：(A)乙酰胆碱受体。我们将延长我们早先的 鱼雷突触后膜生长管状晶体的研究。 我们的第一步是确定五人的身份 跨膜亚单位，离子通路的形状，以及整体 第四系结构。现在，通过将实际发展纳入 显微镜和图像处理我们计划获得对 更高分辨率(8-9A)的结构，足以显示精确的 通道跨膜部分的α-螺旋排列。 我们将使用该地图作为与 打开状态，我们正试图通过快速冻结管子来捕获它 在使用激动剂后的几毫秒内。(B)间隙连接通道。 我们将扩大从大鼠肝脏分离的缝隙连接的早期研究 我们分析了该航道的第四纪结构，并提出了 它打开和关闭的可能机制。为此，我们正在 利用杆状病毒在昆虫细胞中过表达人肝基因 载体和现在正在纯化的重组通道已经获得， 它应该适用于电子结晶学研究 决议。重组通道还被用于三个方面- 维度结晶试验；一些小的微晶体已经 获得。

The broad aim of this research is to understand better the design principles of ion channels and of the mechanisms by which they regulate the flow of ions across cell membranes. The techniques used will include cryoelectron microscopy and crystallographic methods of structure analysis, which we will develop further to obtain higher resolution information. We are also attempting to grow three-dimensional crystals of ion channels suitable for X-ray diffraction studies. The findings from these structural approaches will furnish a three-dimensional framework for relating the extensive data now being obtained by site-directed mutagenesis experiments combined with physiological study of function. Two of the best characterized channels will be investigated as model systems: (a) The acetylcholine receptor. We will extend our earlier studies of the tubular crystals grown from Torpedo postsynaptic membranes. Our first steps have been to determine the identity of the five transmembrane subunits, the shape of the ion pathway, and the overall quaternary structure. Now, by incorporating practical developments in the microscopy and image processing we plan to obtain a description of the structure at higher resolution (8-9A), sufficient to reveal the precise arrangement of alpha-helices in the transmembrane portion of the channel. We will use this map as a basis for comparison with the structure in the open state, which we are attempting to trap by rapidly freezing the tubes within milliseconds of applying the agonist. (b) The gap junction channel. We will extend earlier studies of gap junctions isolated from rat liver in which we analysed the quaternary structure of this channel and suggested a possible mechanism by which it opens and closes. To this end, we are overexpressing the human liver gene in insect cells with a baculovirus vector and are now purifying the recombinant channels have been obtained, which should be suitable for electron crystallographic studies at higher resolution. The recombinant channels are also being used for three- dimensional crystallization trials; some small microcrystals have been obtained.