FUNCTIONAL ANALYSIS OF CELL-CELL CHANNEL PROTEIN

这项研究项目的长期目标是确定分子 细胞-细胞通道的结构并确定细胞-细胞通道的作用 其连接蛋白亚基的不同结构域在细胞周期调控中的作用 频道。鉴于我们对这一问题的理解范围相当有限 细胞-细胞通道的生物学意义亟待学习 更多关于它们的结构和性能的信息。基因克隆的可得性 对于几种缝隙连接蛋白，与强大的非洲爪哇卵母细胞结合 体外合成信使核糖核酸的翻译表达系统 正常的细胞-细胞通道，使下列实验成为可能： 1.确定缝隙连接蛋白与 通过构建各种缺失突变体来获得质膜 可识别的标记肽融合到它们的羧基末端。这项研究将 补充和证实基于绑定的拓扑研究 抗短肽抗体的缝隙连接结构 连接蛋白的片段。 2.通过缺失诱变，确定哪些片段的 连接蛋白32分子是基本的细胞-细胞通道运行所必需的。 这种方法是基于这个实验室最近的实验。 证明了连接蛋白32的大部分羧基末端结构域可以 在不影响频道功能的情况下删除。 3.确定连接蛋白分子的哪些结构域负责 通道的选通属性。这将通过以下方式实现 嵌合连接蛋白的构建结合了细胞- 细胞通道在不同组织中表达。 4.确定其跨膜结构域的结构和功能 连接蛋白32。关于哪些氨基酸参与了 渠道结构的亲水性衬里将通过更换 这些区域中的特定氨基酸。

The longterm goal of this research project is to determine the molecular structure of the cell-cell channel and to determine the roles of the different domains of its connexin protein subunits in the operation of the channel. In view of the rather limited extend of our understanding of the biological significance of cell-cell channels it is imperative to learn more about their structure and properties. The availability of cDNA clones for several gap junction proteins, combined with powerful Xenopus oocyte expression system for the translation of in vitro synthesized mRNA into functional cell-cell channels, makes the following experiments possible: 1. Determine the topology of the gap junction protein in relation to the plasma membrane by constructing various deletion mutants that have an identifiable marker peptide fused to their carboxyl ends. This study will supplement and corroborate topology studies that are based on the binding to gap junction structures of antibodies raised against short peptide segments of the connexin protein. 2. Determine, through deletion mutagenesis, which segments of the connexin32 molecule are essential for basic cell-cell channel operation. This approach is based on recent experiments from this laboratory demonstrating that most of the carboxyl terminal domain of connexin32 can be deleted without affecting channel function. 3. Determine which domains of the connexin molecule is/are responsible for the gating properties of the channel. This will be accomplished through the construction of chimeric connexins combining the properties of cell- cell channels expressed in different tissues. 4. Determine the structure and function of the transmembrane domains of connexin32. The question as to which amino acids are participating in the hydrophilic lining of the channel structure will be addressed by replacing specific amino acids in these domains.