3-D DESIGN AND ACTION OF CELL MEMBRANE CHANNELS

本研究的目的是为了更好地理解设计 细胞膜通道的原理以及 它们调节跨膜的离子通量。 所使用的技术将 包括低温电子显微镜和晶体学方法， 三维结构分析;系统的努力将是 继续获得适合于X射线衍射的晶体 问题研究 研究结果将提供三维框架 为了将现在获得的大量数据与 生物化学、药理学、遗传学和生理学 两个最好的特征通道将被研究为 模型系统：（a）乙酰胆碱受体。 我们计划扩大 我们对生长的管状晶体进行了电子显微镜分析 分离的突触后膜 我们的第一步是 以低分辨率确定受体的结构，以及 以确定各个子单元的身份。 现在通过 结合显微镜和数据的实际发展， 处理，我们将分析单个子单元的详细信息， 更精细的尺度，并评估不同的构象状态。 初步的三维地图显示 静息状态和脱敏状态之间的差异。 试点 已经开始了研究开放状态的实验。 （B） 差距连接通道。 我们计划进一步调查 结构和功能的差距结沟道，使用 从大鼠肝脏分离的斑块和广泛的膜片 最近通过过表达编码人 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，Ca2 + 诱导构象变化，这表明了一种合理的机制 通道通过其打开和关闭。 现在的工作旨在描述 这些部分有助于促进 构象变化 氨基酸序列的特征 以紧密配合结构细节，并导致一个可测试的 多肽链折叠的模型。 跨膜 一部分似乎是一个四阿尔法螺旋束，我们计划 直接通过改进低温系统来解决， 电子显微镜技术和利用衍生的膜 从酵母中分离出来

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.