3D DESIGN AND ACTION OF CELL MEMBRANE CHANNELS

描述：本研究的广泛目的是为了更好地了解 离子通道的设计原则和离子通道的机制 调节跨细胞膜的离子运输。所使用的技术将 包括确定结构电子结晶学方法， 它将被进一步发展来分析快速构象变化 并获得更高分辨率的信息。申请者还将 生长适合于离子通道的三维晶体的尝试 X射线衍射研究。从这些结构方法中得到的发现 将为关联大量数据提供三维框架 现在是通过定点突变实验结合起来获得的 具有电生理研究的功能，并应提供直接 洞察渠道是如何工作的。 将调查三个有特色的渠道：(A) 乙酰胆碱受体。安文博士将扩展他的研究范围 从鱼雷突触后膜生长的管状晶体， 现在已经以9A的分辨率确定了受体结构 以封闭和(最近)开放渠道的形式。他会的 在9A分辨率下确定受体的结构 脱敏状态，以推导出对 在这个分辨率下发生的构象变化。同时， 随着显微镜和图像处理技术的进一步发展，他将 努力获得更详细的结构描述，请参见 闭合通道形式(分辨率：4-5A)。(B)缝隙连接处 频道。安文博士将扩展早期对缝隙连接的研究 从大鼠肝脏中分离出来，在其中他分析了 并建议了一种可能的机制，它通过这种机制打开和 关门了。图像处理方法的最新发展，应用于 二维表，现在使他能够探索他们的三维- 9 A分辨率的维度结构，而不是18 A分辨率 是以前做过的。作为一种替代方法，他还 利用杆状病毒在昆虫细胞中过表达人肝基因 载体，现在正在纯化重组通道，使其在 毫克量。通道的广泛水晶片具有 最终可能适用于结晶学 以更高的分辨率进行研究。(C)电压门控钾通道。一个 电压门控振荡器钾通道的研究已经开始 理解采取的结构性变化的长期目标 响应电压变化的位置，类似于实验 在配基门控离子通道上进行。申请人是 想知道电压门控通道是否打开和关闭 类似或完全无关的机制。

DESCRIPTION: The broad aim of this research is to understand better the design principles of ion channels and of the mechanisms by which they regulate ion transport across cell membranes. The techniques used will include electron crystallographic methods of structure determination, which will be developed further to analyze rapid conformational changes and to obtain higher resolution information. The applicant will also attempt to grow three-dimensional crystals of ion channels suitable for X-ray diffraction studies. The finding from these structural approaches will furnish three-dimensional frameworks for relating the extensive data now being obtained by site-directed mutagenesis experiments combined with electro-physiological study of function, and should provide direct insight into how the channels work. Three well characterized channels will be investigated: (a) The acetylcholine receptor. Dr. Unwin will extend his studies of the tubular crystals grown from Torpedo postsynaptic membranes, from which the receptor structure has now been determined at a resolution of 9 A in both the closed- and (very recently) open-channel forms. He will determine the structure of the receptor at 9 A resolution in the desensitized state, to derive a complete description of the conformational changes occurring at this resolution. At the same time, by further developments in the microscopy and image processing, he will work towards obtaining a more detailed description of the structure in the closed-channel form (resolution: 4-5 A). (b) The gap junction channel. Dr. Unwin will extend earlier studies of gap junctions isolated from rat liver in which he analyzed the quaternary structure of this channel and suggested a possible mechanisms by which it opens and closes. Recent developments in image processing methods, applied to two-dimensional sheets, are enabling him now to explore their three- dimensional structures at 9 A resolution, rather than 18 A resolution as was done previously. As an alternative approach, he is also overexpressing the human liver gene in insect cells with a baculovirus vector and is now purifying the recombinant channels to homogeneity in milligram quantities. Extensive crystalline sheets of the channels have been obtained, which may eventually be suitable for crystallographic studies at higher resolution. (c) Voltage-gated potassium channels. A study of voltage-gated Shaker potassium channels has been initiated with the long-term goal of understanding the structural changes that take place in response to changes in voltage, in analogy with the experiments being performed on the ligand-gated ion channels. The applicant is interested to know if the voltage-gated channels open and close by similar, or completely unrelated, mechanisms.