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 离子通道的原理和它们调节离子通道的机制 离子穿过细胞膜的流动。 所使用的技术将包括 低温电子显微术和结构分析的晶体学方法， 我们将进一步开发以获得更高分辨率的信息。 我们 也在尝试生长离子通道的三维晶体 适用于X射线衍射研究。 这些结构性的发现 方法将提供一个三维框架， 通过定点突变实验， 结合生理功能研究。 两个最好的特征通道将作为模型进行研究 系统：（a）乙酰胆碱受体。 我们将继续我们的早期 研究从电鳐突触后膜生长的管状晶体。 我们的第一步是确定这五个人的身份 跨膜亚基，离子通路的形状，以及整体的 四级结构 现在，通过将实际发展纳入 显微镜和图像处理，我们计划获得的描述， 结构在更高的分辨率（8-9A），足以揭示精确的 通道的跨膜部分中的α-螺旋的排列。 我们将使用此图作为与 开放状态，我们试图通过快速冻结管道来捕获 在注射激动剂的几毫秒内 (b)差距连接通道。 我们将扩展早期的研究，从大鼠肝脏分离缝隙连接， 我们分析了这个通道的四级结构，并提出了一个 可能的机制，通过它打开和关闭。 为此，我们 用杆状病毒在昆虫细胞中过表达人肝基因 载体和现在纯化的重组通道已经获得， 这应该是适合于电子晶体学研究在较高的 分辨率 重组通道也被用于三个- 三维结晶试验;一些小的微晶已经 得到了

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.