Heteromeric connexons formed by the lens connexins, connexin43 and connexin56

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 betwee n 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 band s of Cx56 corresponding to phosphorylated forms were detected by immunoblot ting in NRK-Cx56 cells. Immunofluorescence studies showed co-localization o f Cx56 with Cx43 in the perinuclear region and at appositional membranes, C onnexin hexamers in NRK-Cx56 cells contained both Cx43 and Cx56 as demonstr ated by sedimentation through sucrose gradients. Immunoprecipitation of Cx5 6 from sucrose gradient fractions resulted in co-precipitation of Cx43 from NRK-Cx56 cells suggesting the presence of relatively stable interactions b etween the two connexins, Double whole-cell patch-clamp experiments showed that the voltage-dependence of G(min) 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 heterom eric 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.