Martin P E, Errington R J, Evans W H
Wales Heart Research Institute and Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK.
Cell Commun Adhes. 2001;8(4-6):243-8. doi: 10.3109/15419060109080731.
The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.
利用表达连接蛋白（Cx）26、32和43的哺乳动物细胞研究了间隙连接通道的组装，其中这些连接蛋白的羧基末端与绿色、黄色或青色荧光蛋白（GFP、YFP、CFP）融合。布雷菲德菌素A处理破坏了Cx32 - CFP和43 - GFP向间隙连接的细胞内靶向作用，并导致间隙连接细胞间通讯严重丧失，表现为细胞间染料转移率降低。用诺考达唑处理表达Cx43 - GFP的细胞，其向间隙连接的靶向作用和染料转移正常。因此，Cx32和43似乎是通过经典分泌途径转运并组装到间隙连接中的。相比之下，我们发现Cx26 - GFP组装成功能性间隙连接相对不受布雷菲德菌素A处理细胞的影响，但对诺考达唑处理极为敏感。Cx26 - YFP和Cx32 - CFP的共表达显示了不同的细胞内分布，在布雷菲德菌素A存在的情况下这种分布更加明显，这些细胞中的间隙连接主要由Cx26 - YFP构成。区分Cx32与Cx26的第一个跨膜结构域中的位点特异性突变（Cx32 128L）导致其具有Cx26的转运特性及其异常的翻译后膜整合特征。结果表明存在多种细胞内连接蛋白转运途径，并为调节间隙连接的连接蛋白组成从而调节细胞间信号传导特异性提供了进一步的机制。
