Synthesis and assembly of human beta 1 gap junctions in BHK cells by DNA transfection with the human beta 1 cDNA.
Synthesis and assembly of human beta 1 gap junctions in BHK cells by DNA transfection with the human beta 1 cDNA.
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发表时间：
1995-12
影响因子：
4
通讯作者：
Nalin M. Kumar;Daniel S. Friend;N. Gilula
中科院分区：
文献类型：
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作者：
Nalin M. Kumar;Daniel S. Friend;N. Gilula
间隙连接通讯在许多生理过程中都很重要，包括生长控制、模式和细胞间活动的同步。由于其丰度较低，对其合成和组装的研究一直很困难。为了克服这一限制，用人β 1 （Cx32）连接蛋白cDNA构建物转染了幼鼠肾细胞（BHK）。在小鼠金属硫蛋白启动子的控制下表达，该启动子可被重金属诱导。对转染后的细胞进行DNA、RNA和蛋白质分析，并通过刮片加载检测功能通道。功能β 1连接蛋白仅在以正确方向（β 1- bhk）转染β 1连接蛋白cDNA的细胞中检测到。光镜免疫细胞化学分析表明，β 1连接蛋白抗原定位于质膜和几个细胞内区室。薄层电子显微镜显示细胞之间（在细胞表面）、内质网（ER）和核膜中存在广泛的双膜间隙连接。冷冻断裂分析证实了这种不寻常的间隙连接蛋白在细胞内的分布，在所有这些膜的断裂面上发现了间隙连接斑块特征的大颗粒聚集体。内质网中间隙连接粒子聚集体的存在表明连接蛋白的寡聚化可能发生在其合成位点。此外，在细胞膜内组装成双膜连接结构的过程可能受到连接蛋白浓度的驱动。
Gap junctional communication is important in many physiological processes, including growth control, patterning, and the synchronization of cell-to-cell activities. It has been difficult to study the synthesis and assembly of gap junctions due to their low abundance. To overcome this limitation, baby hamster kidney cells (BHK) have been transfected with a human beta 1 (Cx32) connexin cDNA construct. Expression was placed under the control of the mouse metallothionein promoter that can be induced by heavy metals. The transfected cells were characterized by DNA, RNA and protein analysis, as well as by scrape loading to detect functional channels. Functional beta 1 connexin was detected only in cells transfected with beta 1 connexin cDNA in the correct orientation (beta 1-BHK). Analysis of the cells by light microscopic immunocytochemistry indicated that beta 1 connexin antigen was localized to the plasma membrane and to several intracellular compartments. Characterization with thin section electron microscopy revealed extensive areas of assembled double membrane gap junctions between cells (on the cell surface), in the endoplasmic reticulum (ER), and the nuclear envelope. This unusual intracellular distribution for assembled gap junction protein was confirmed by freeze fracture analysis, which revealed large particle aggregates, characteristic of gap junction plaques, on the fracture faces of all these membranes. The presence of gap junction particle aggregates in the ER suggests that the oligomerization of connexin can occur at its site of synthesis. Further, the process of assembly into double membrane junction structures in intracellular membranes may be driven by connexin protein concentration.