Cell-free synthesis and assembly of connexins into functional gap junction membrane channels

Abstract

Several different gap junction channel subunit isotypes, known as connexins, were synthesized in a cell-free translation system supplemented with microsomal membranes to study the mechanisms involved in gap junction channel assembly. Previous results indicated that the connexins were synthesized as membrane proteins with their relevant transmembrane topology. An integrated biochemical and biophysical analysis indicated that the connexins assembled specifically with other connexin subunits. No interactions were detected between connexin subunits and other co-translated transmembrane proteins. The connexins that were integrated into microsomal vesicles assembled into homo- and hetero-oligomeric structures with hydrodynamic properties of a 9S particle, consistent with the properties reported for hexameric gap junction connexons derived from gap junctions in vivo. Further, cell-free assembled homo-oligomeric connexons composed of beta1 or beta2 connexin were reconstituted 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 evidence that the synthesis and assembly of a gap junction connexon can take place in microsomal membranes. Finally, the cell-free system has been used to investigate the properties of alpha1, beta1 and beta2 connexin 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 connexin isotypes may be located in the N-terminal sequence of the connexins.

Gating Connexin 43 Channels Reconstituted in Lipid Vesicles by Mitogen-activated Protein Kinase Phosphorylation

“…Two distinct Cx43 protein peaks corresponding to 5 S and 9 S particles were detected. Previous studies (39) have demonstrated that the 5 S Cx43 population contributes the monomeric form of the protein, whereas the 9 S species represents the hexameric form. Our data showed that only one-half of the purified Cx43 protein was assembled into connexons.…”

Mutation R184Q of connexin 26 in hearing loss patients has a dominant-negative effect on connexin 26 and connexin 30

“…7 Other studies have shown that Cxs can assemble into functional hexameric connexons in the endoplasmic reticulum (ER) membrane. 8 Subcellular fractionation studies and immunocolocalization analyses suggest that Cxs reach the plasma membrane by passing through the Golgi apparatus. [9][10][11] Twenty-one genes have been identified in the human genome coding for connexin proteins.…”

A Carboxyl Terminal Domain of Connexin43 Is Critical for Gap Junction Plaque Formation but not for Homo- or Hetero-Oligomerization

“…Oligomerization may involve all identical subunits (to form homomeric connexons) or various combinations of compatible connexins (to form heteromeric connexons). The cellular process of connexin oligomerization is beginning to be elucidated (1,6,8,18). We and others have identified a number of compatible or incompatible partners for the formation of heteromeric connexons (3,4,5,10,11,15).…”