FUNCTIONAL ANALYSIS OF CELL-CELL CHANNEL PROTEIN

这个研究项目的长期目标是确定 细胞-细胞通道的结构，并确定 连接蛋白亚基的不同结构域参与了 频道 鉴于我们对这一问题的了解相当有限， 细胞-细胞通道的生物学意义，必须学习 更多关于它们的结构和性质。 cDNA克隆的可用性 几种缝隙连接蛋白，结合强大的非洲爪蟾卵母细胞 用于将体外合成的mRNA翻译成 功能性细胞-细胞通道，使得以下实验成为可能： 1. 确定差距连接蛋白相对于 通过构建各种缺失突变体， 与其羧基末端融合的可识别标记肽。 本研究将 补充和证实基于绑定的拓扑研究 针对短肽产生的抗体的间隙连接结构 连接蛋白的片段。 2. 通过缺失突变确定， 连接蛋白32分子对于基本细胞-细胞通道操作是必需的。 这种方法是基于该实验室最近的实验 这表明连接蛋白32的大多数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 通道的门控特性。 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 通道结构的亲水衬里将通过替换 在这些领域的特定氨基酸。

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.