Berthoud V M, Montegna E A, Atal N, Aithal N H, Brink P R, Beyer E C
Department of Pediatrics, University of Chicago, IL 60637, USA.
Eur J Cell Biol. 2001 Jan;80(1):11-9. doi: 10.1078/0171-9335-00132.
In the eye lens, three connexins have been detected in epithelial cells and bow region/differentiating fiber cells, suggesting the possible formation of heteromeric gap junction channels. To study possible interactions between Cx56 and Cx43, we stably transfected a normal rat kidney cell line (NRK) that expresses Cx43 with Cx56 (NRK-Cx56). Similar to the lens, several bands of Cx56 corresponding to phosphorylated forms were detected by immunoblotting in NRK-Cx56 cells. Immunofluorescence studies showed co-localization of Cx56 with Cx43 in the perinuclear region and at appositional membranes. Connexin hexamers in NRK-Cx56 cells contained both Cx43 and Cx56 as demonstrated by sedimentation through sucrose gradients. Immunoprecipitation of Cx56 from sucrose gradient fractions resulted in co-precipitation of Cx43 from NRK-Cx56 cells suggesting the presence of relatively stable interactions between the two connexins. Double whole-cell patch-clamp experiments showed that the voltage-dependence of Gmin in NRK-Cx56 cells differed from that in NRK cells. Moreover, stable interactions between Cx43 and Cx56 were also demonstrated in the embryonic chicken lens by co-precipitation of Cx43 in Cx56 immunoprecipitates. These data suggest that Cx43 and Cx56 form heteromeric connexons in NRK-Cx56 cells as well as in the lens in vivo leading to differences in channel properties which might contribute to the variations in gap junctional intercellular communication observed in different regions of the lens.
在眼晶状体中，已在上皮细胞以及Bow区域/分化的纤维细胞中检测到三种连接蛋白，这表明可能形成了异源间隙连接通道。为了研究Cx56和Cx43之间可能的相互作用，我们用Cx56稳定转染了表达Cx43的正常大鼠肾细胞系（NRK）（NRK-Cx56）。与晶状体类似，通过免疫印迹在NRK-Cx56细胞中检测到了几条与磷酸化形式相对应的Cx56条带。免疫荧光研究显示Cx56与Cx43在核周区域和相邻细胞膜处共定位。通过蔗糖梯度沉降证明，NRK-Cx56细胞中的连接蛋白六聚体同时包含Cx43和Cx56。从蔗糖梯度级分中对Cx56进行免疫沉淀导致NRK-Cx56细胞中的Cx43共沉淀，这表明两种连接蛋白之间存在相对稳定的相互作用。双全细胞膜片钳实验表明，NRK-Cx56细胞中Gmin的电压依赖性与NRK细胞中的不同。此外，通过在Cx56免疫沉淀物中Cx43的共沉淀，也证明了胚胎鸡晶状体中Cx43和Cx56之间存在稳定的相互作用。这些数据表明，Cx43和Cx56在NRK-Cx56细胞以及体内晶状体中形成异源连接子，导致通道特性的差异，这可能有助于解释在晶状体不同区域观察到的间隙连接细胞间通讯的变化。
