Multimeric connexin interactions prior to the trans-Golgi network

Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin co nstruct, consisting of bacterial beta -galactosidase fused to the C terminu s of connexin43 (Cx43/beta -gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/beta -gal is retained in a perinuclear compartment and inhi bits Cx43 transport to the cell surface. The intracellular connexin pool in duced by Cx43/beta -gal colocalized with a medial Golgi apparatus marker an d was readily disassembled by treatment with brefeldin A. This was unexpect ed, since previous studies indicated that Cx43 assembly into hexameric hemi channels occurs in the trans-Golgi network (TGN) and is sensitive to brefel din A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta -gal were assembled into a subhexameric complex. Cx43/beta -g al also specifically interacted with Cx46, but not Cx32, consistent with th e ability of Cx43/beta -gal to simultaneously inhibit multiple connexins. W e confirmed that interactions between Cx43/beta -gal and Cx46 reflect the a bility of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveo lar epithelial cells, which express both connexins. In contrast, ROS osteob lastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx 46 heteromers. Thus, cells have the capacity to regulate whether or not com patible connexins intermix.