Connexin45 interacts with zonula occludens-1 and connexin43 in osteoblastic cells

The relative expression of connexin43 and connexin45 modulates gap junction al communication and production of bone matrix proteins in osteoblastic cel ls, It is likely that changes in gap junction permeability are determined b y the interaction between these two proteins. Cx43 interacts with ZO-1, whi ch may be involved in trafficking of Cx43 or facilitating interactions betw een Cx43 and other proteins. In this study we sought to identify proteins t hat associate with Cx45 by coprecipitation in non-denaturing conditions. Cx 45 was isolated with a 220-kDa protein that we identified as ZO-1. Under th e same conditions, Cx43 also was isolated with anti-Cx45 antiserum from Cx4 5-transfected ROS cells (ROS/Cx45 cells). Cx43 antiserum could also copreci pitate ZO-1 in the transfected and untransfected ROS cells. Double label im munofluorescence studies showed that ZO-1, Cx43, and Cx45 colocalized at ap positional membranes in ROS/Cx45 cells suggesting that all three proteins a re normally associated in the cells, Additionally, we found that in vitro t ranslated ZO-1 binds to the carboxyl-terminal of Cx45 indicating that there is a direct interaction between the carboxyl-terminal of Cx45 and ZO-1. Th ese studies demonstrate that ZO-1 interacts with Cx45 as well as with Cx43, and suggest that the interaction of connexins with ZO-1 may play a role in regulating the composition of the gap junction and may modulate connexin-c onnexin interactions.