FUNCTIONAL ANALYSIS OF CELL-CELL CHANNEL PROTEIN

这个研究项目的长期目标是确定分子

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.