MOLECULAR CLONING OF CARDIAC GAP JUNCTION ION CHANNELS

缝隙连接介导的细胞间通讯对 心肌细胞之间兴奋的同步传播。我提议 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，我已经有了 大鼠心脏缝隙相关基因的克隆与鉴定 连接蛋白。心脏缝隙连接的主要序列为 测定并与肝脏进行比较。针对特定蛋白质的抗体 将产生结构域，并用于通过免疫细胞化学确定 方法：通道蛋白的跨膜定位。航道 用克隆的cDNA来研究蛋白质的形成能力。 在体外产生分子上纯的心脏缝隙连接蛋白。这种蛋白质 将通过翻译合成的蛋白质进入膜囊泡 在狗胰腺微粒体存在的情况下，然后融合成 平面脂质双层膜的离子选择性和门控性能 可以精确地确定通道。我将尝试开发一种 制度所在地方建立差距的要求 可以检查连接介导的细胞间通讯。非洲爪哇 卵母细胞将被注射合成的RNA。它的合成、加工、 将检查缝隙连接蛋白的膜插入和组装。 两对相似注射的卵母细胞形成功能间隙的能力 交界处将进行评估。这些研究将提供分子基础 为了理解心脏细胞之间的同步性，以及 伴随心脏缺血的同步化模式的发病机制 和再灌注期。

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.