TRANSFECTED CONNEXIN45 ALTERS GAP JUNCTION PERMEABILITY IN CELLS EXPRESSING ENDOGENOUS CONNEXIN43

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), whil e connexin45 (Cx45) channels do not. We transfected full-length or tru ncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, whic h expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected wit h Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showe d 30-60% of the gap junctional conductance exhibited by ROS cells. Int ercellular transfer of three negatively charged fluorescent reporter m olecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to >20 cells. Transfer of calcein (MW = 623 D) was als o reduced by similar to 50% in ROS/Cx45 cells, but passage of hydroxyc oumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellula r coupling between cells expressing Cx43, most likely the result of di rect interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx4 5 had different effects in ROS cells, consistent with a role of the ca rboxyl-terminal domain of Cx45 in determining gap junction per meabili ty or interactions between connexins. These data suggest that coexpres sion of multiple connexins may enable cells to achieve forms of interc ellular communication that cannot be attained by expression of a singl e connexin.