CELL-FREE SYNTHESIS AND ASSEMBLY OF CONNEXINS INTO FUNCTIONAL GAP JUNCTION MEMBRANE CHANNELS

Several different gap junction channel subunit isotypes, known as conn exins, were synthesized in a cell-free translation system supplemented with microsomal membranes to study the mechanisms involved in gap jun ction channel assembly. Previous results indicated that the connexins were synthesized as membrane proteins with their relevant transmembran e topology, An integrated biochemical and biophysical analysis indicat ed that the connexins assembled specifically with other connexin subun its, No interactions were detected between connexin subunits and other co-translated transmembrane proteins, The connexins that were integra ted into microsomal vesicles assembled into homo- and hetero-oligomeri c structures with hydrodynamic properties of a 9S particle, consistent with the properties reported for hexameric gap junction connexons der ived from gap junctions in vivo. Further, cell-free assembled homo-oli gomeric connexons composed of beta(1) or beta(2) connexin were reconst ituted into synthetic lipid bilayers. Single channel conductances were recorded from these bilayers that were similar to those measured for these connexons produced in vivo. Thus, this is the first direct evide nce that the synthesis and assembly of a gap junction connexon can tak e place in microsomal membranes. Finally, the cell-free system has bee n used to investigate the properties of al, beta(1) and beta(2) connex in to assemble into hetero-oligomers. Evidence has been obtained for a selective interaction between individual connexin isotypes and that a signal determining the potential hetero-oligomeric combinations of co nnexin isotypes may be located in the N-terminal sequence of the conne xins.