MOLECULAR CLONING OF CARDIAC GAP JUNCTION ION CHANNELS

由缝隙连接介导的细胞间通讯是细胞间通讯的基础。 心肌细胞之间兴奋的同步传播。 我建议 研究心脏缝隙连接通道的生物化学和生物物理学。 我将使用大鼠肝脏缝隙连接蛋白的cDNA， 克隆和鉴定，作为探针分离大鼠心脏间隙的cDNA 连接蛋白 心脏缝隙连接的主要序列将是 测定并与肝脏进行比较。 特定蛋白质的特异性抗体 将产生结构域并用于通过免疫细胞化学 方法，通道蛋白的跨膜取向。 信道 蛋白质的形成能力将通过使用克隆的cDNA来研究， 在体外产生分子纯的心脏缝隙连接蛋白。 蛋白质 将通过翻译合成的 mRNA在狗胰腺微粒体的存在下，然后融合到 平面脂质双层，其中的离子选择性和门控特性 可以精确地确定信道。 我将尝试开发一个 系统在哪里建立差距的要求 可以检测连接介导的细胞间通讯。 爪蟾 卵母细胞将注射合成RNA。 合成，加工， 将检查间隙连接蛋白的膜插入和组装。 相似注射的卵母细胞对形成功能间隙的能力 将对交叉口进行评估。 这些研究将提供分子基础 对于理解心脏细胞之间的同步， 心肌缺血同步化模式的发病机制 和再灌注发作。

Intercellular communication mediated by gap junctions is essential for the synchronized spread of excitation between myocardial cells. I propose to study the biochemistry and biophysics of the cardiac gap junction channel. I will use cDNA for rat liver gap junction protein, which I have already cloned and characterized, as a probe to isolate cDNA for rat cardiac gap junction protein. The primary sequence of the heart gap junction will be determined and compared to liver. Antibodies specific to defined protein domains will be produced and used to determine, by immunocytochemical methods, the transmembrane orientation of the channel protein. The channel forming ability of the protein will be studied by using the cloned cDNA to produce molecularly pure heart gap junction protein in vitro. The protein will be incorporated into membrane vesicles by translation of synthetic mRNA in the presence of dog pancreatic microsomes and then fused into planar lipid bilayers where the ion selectivity and gating properties of the channel can be precisely determined. I will attempt to develop a system where the requirements for the establishment of gap junction-mediated intercellular communication can be examined. Xenopus oocytes will be injected with synthetic RNA. The synthesis, processing, membrane insertion and assembly of gap junction protein will be examined. The ability of pairs of similarly injected oocytes to form functional gap junctions will be assessed. These studies will provide a molecular basis for the understanding of synchronization between heart cells, and the pathogenesis in synchronization patterns which accompany cardiac ischemic and reperfusion episodes.