3-D DESIGN AND ACTION OF CELL MEMBRANE CHANNELS

这项研究的目的是为了更好地理解设计 细胞膜通道的原理及其作用机制 它们调节跨膜的离子流量。所使用的技术将 包括低温电子显微镜和结晶学方法 三维结构分析；将系统地努力 继续获得适合x射线衍射的晶体 学习。这些发现将提供三维的框架 将现在从以下来源获得的大量数据联系起来 生化、药理学、遗传学和生理学 将对两个特征最好的渠道进行调查 模型系统：(A)乙酰胆碱受体。我们计划延长 我们生长的管状晶体的电子显微镜分析 来自分离的突触后膜。我们的第一步是 在低分辨率下确定受体的结构，以及 以确定各个亚基的身份。现在，由 结合显微镜和数据方面的实际发展 处理过程中，我们将分析单个亚基在 更精细的尺度，并评估不同的构象状态。 已经获得了初步的三维地图，揭示了 静息状态和脱敏状态之间的差异。引航员 已经开始了实验来研究这种开放状态。(B) 缝隙结通道。我们计划进一步调查 间隙结通道的结构和功能，使用 从大鼠肝脏分离的斑块和广泛的膜片 最近通过过度表达编码人类的 酵母中的肝多肽。我们的第一步是确定 这种蛋白质的四级结构。我们还发现，钙离子 导致构象变化，暗示了一种合理的机制 通道通过其打开和关闭。现在的工作目标是描述 那些有助于促进 构象变化。出现了氨基酸序列的特征 为了与结构细节紧密匹配，并导致了可测试 多肽链折叠的模型。跨膜的 部分看起来是一个四个阿尔法螺旋束，我们计划 通过在冷冻机中加入改进来直接解决- 电子显微镜技术及其衍生膜的利用 从酵母中提取。

The purpose of this research is to understand better the design principles of cell membrane channels and the mechanisms by which they regulate ion flux across membranes. The techniques used will include cryo-electron microscopy and crystallographic methods of three-dimensional structure analysis; systematic efforts will be continued to obtain crystals suitable for x-ray diffraction studies. The findings will furnish three-dimensional framework for relating the extensive data now being obtained from biochemical, pharmacological, genetic and physiological Two of the best characterized channels will be investigated as model systems: (a) The acetylcholine receptor. We plan to extend our electron microscopical analyses of the tubular crystals grown from isolated postsynaptic membranes. Our first steps have been to determine the structure of the receptor at low resolution, and to establish the identity of the individual subunits. Now, by incorporating practical developments in the microscopy and the data processing, we will analyze details of individual subunits on a finer scale, and evaluate different conformational states. Preliminary three-dimensional maps have been obtained revealing differences between resting and desensitized states. Pilot experiments have been initiated to investigate the open state. (b) The gap junction channel. We plan to investigate further the structure and function of the gap junction channel, using the plaques isolated from rat liver and the extensive membrane sheets recently derived by over-expression of the cDNA encoding the human liver polypeptide in yeast. Our first step has been to determine the quaternary structure of this protein. We also found that Ca2+ induces a conformational change, suggesting a plausible mechanism by which the channel opens and closes. Work now aims to describe those parts which are instrumental in facilitating the conformational change. Features of the amino acid sequence appear to fit closely the structural details and have led to a testable model for the folding of the polypeptide chain. The transmembrane portion appears to be a four alpha-helical bundle, which we plan to resolve directly by incorporating improvements in the cryo- electron microscopy technology and utilizing the membranes derived from the yeast.