STRUCTURE OF THE NICOTINIC ACETYLCHOLINE RECEPTOR

本应用程序的长期目标是了解 AChR的结构特征，并将这些结构与 受体的功能。 AChR的结构可以作为一个范例 为配体门控离子通道的结构提供了基础 了解这类蛋白质的功能。 的 AChR上ACh结合位点的结构将通过以下方法进行研究： 光亲和标记，以确定相应的贡献， 结合位点的亚基。 TC将用作光亲和标记; γ-和 与TC反应的δ亚基将通过分离 标记片段和通过N-末端鉴定标记残基 氨基酸测序 TC将用光反应基团衍生化 以增强光反应性，然后还用于表征结合 网站以同样的方式。 AChR亚基初级的拓扑结构 序列将通过标记暴露在 AChR的细胞外或细胞内表面。 标记位点 将通过分离标记的片段并鉴定 标记的残留物。 一个假定的细胞内暴露的 片段alpha 156-179将被专门评估。 化学交叉- 链接将用于定义 亚基和亚基内的单个氨基酸。 附近的残留物 乙酰胆碱结合位点将通过特异性连接光活化的 交联剂与ACh结合位点附近的二硫化物交联。

The long-term objectives of this application are to understand the structural features of the AChR and to relate these structures to the function of the receptor. The structure of the AChR serves as a paradigm for the structure of ligand gated ion channels and thus provides a basis for an understanding of how this class of proteins function. The structure of the ACh binding sites on the AChR will be investigated by photoaffinity labelling to determine the respective contribution of the subunits to the binding sites. TC will be used as a photoaffinity label; the sites of the gamma- and delta-subunits that react with TC will be defined through isolation of labelled fragments and identification of labelled residues by N-terminal amino acid sequencing. TC will be derivatized with photoreactive groups to enhance photoreactivity and then also used to characterize the binding sites in the same manner. The topology of the AChR subunit primary sequence will be analyzed by labelling of protein exposed on the extracellular or intracellular surface of the AChR. The labelled sites will be identified by isolation of labelled fragments and identification of labelled residues. The location of a putative intracellularly exposed fragment alpha 156-179 will be evaluated specifically. Chemical cross- linking will be used to define the nearest-neighbor relationships of the subunits and of individual amino acids within subunits. Residues near the ACh binding sites will be probed by specifically attaching photoactivated cross-linkers to the disulfide near the ACh binding site.