Jiang J X, White T W, Goodenough D A, Paul D L
Department of Cell Biology, Neurobiology Harvard Medical School, Boston 02115.
Mol Biol Cell. 1994 Mar;5(3):363-73. doi: 10.1091/mbc.5.3.363.
The avian lens is an ideal system to study gap junctional intercellular communication in development and homeostasis. The lens is experimentally more accessible in the developing chick embryo than in other organisms, and chick lens cells differentiate well in primary cultures. However, only two members of the connexin gene family have been identified in the avian lens, whereas three are known in the mammalian system. We report here the molecular cloning and characterization of the third lens connexin, chick connexin45.6 (ChCx45.6), a protein with a predicted molecular mass of 45.6 kDa. ChCx45.6 was encoded by a single copy gene and was expressed specifically in the lens. There were two mRNA species of 6.4 kilobase (kb) and 9.4 kb in length. ChCx45.6 was a functional connexin protein, because expression in Xenopus oocyte pairs resulted in the development of high levels of conductance with a characteristic voltage sensitivity. Antisera were raised against ChCx45.6 and chick connexin56 (ChCx56), another avian lens-specific connexin, permitting the examination of the distribution of both proteins. Immunofluorescence localization showed that both ChCx45.6 and ChCx56 were abundant in lens fibers. Treatment of lens membranes with alkaline phosphatase resulted in electrophoretic mobility shifts, demonstrating that both ChCx45.6 and ChCx56 were phosphoproteins in vivo.
禽类晶状体是研究发育和内环境稳定过程中缝隙连接细胞间通讯的理想系统。与其他生物体相比，发育中的鸡胚晶状体在实验上更容易获取，并且鸡晶状体细胞在原代培养中分化良好。然而，在禽类晶状体中仅鉴定出连接蛋白基因家族的两个成员，而在哺乳动物系统中已知有三个。我们在此报告第三个晶状体连接蛋白——鸡连接蛋白45.6（ChCx45.6）的分子克隆和特性，该蛋白预测分子量为45.6 kDa。ChCx45.6由单拷贝基因编码，且在晶状体中特异性表达。有两种长度分别为6.4千碱基（kb）和9.4 kb的mRNA。ChCx45.6是一种功能性连接蛋白，因为在非洲爪蟾卵母细胞对中表达会导致具有特征电压敏感性的高水平电导的形成。制备了针对ChCx45.6和另一种禽类晶状体特异性连接蛋白——鸡连接蛋白56（ChCx56）的抗血清，从而能够检测这两种蛋白的分布。免疫荧光定位显示ChCx45.6和ChCx56在晶状体纤维中都很丰富。用碱性磷酸酶处理晶状体膜导致电泳迁移率发生变化，表明ChCx45.6和ChCx56在体内都是磷蛋白。
