Synthesis and assembly of connexins in vitro into homomeric and heteromeric functional gap junction hemichannels

The biogenesis of connexins and their assembly into functional gap junction hemichannels (connexons) was studied with the use of a cell-free transcrip tion/translation system. Velocity sedimentation on sucrose gradients showed that a small proportion of connexin (Cx) 26 and Cx32 that were co-translat ionally translocated into microsomes were oligomers of Cx26 and Cx32. Chemi cal cross-linking studies showed that these corresponded to hexameric conne xons. Reconstitution of connexons synthesized in vitro into liposomes induc ed permeability properties consistent with the view that open gap junction hemichannels were produced. By using an immunoprecipitation approach, a sim ultaneous translation of Cx26 and Cx32 incorporated into microsomes resulte d in homomeric connexons. However, supplementation of the translation syste m in vitro with liver Golgi membranes produced heteromeric connexons constr ucted of Cx32 and Cx26, and also resulted in an increased oligomerization e specially of Cx32. All of the connexins analysed were inserted co-translati onally into canine pancreatic microsomal membranes. In addition, Cx26 and C x43, but not Cx32, were also inserted into microsomal membranes post-transl ationally. Analysis of various connexin constructs in which the cytoplasmic carboxy tails were transposed, the cytoplasmic tail of Cx43 was truncated or a reporter protein, aequorin, was attached to the C-terminus showed that tail length was not the major determinant of the post-translational membra ne insertion of connexins.