Gap junctions consist of clusters of intercellular channels, which enable direct cell-to-cell communication and adhesion in animals. Whereas deuterostomes, including all vertebrates, use members of the connexin and pannexin multiprotein families to assemble gap junction channels, protostomes such as Drosophila and Caenorhabditis elegans use members of the innexin protein family. The molecular composition of innexin-containing gap junctions and the functional significance of innexin oligomerization for development are largely unknown. Here, we report that heteromerization of Drosophila innexins 2 and 3 is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins colocalize in epithelial cell membranes. Innexin3 is mislocalized to the cytoplasm in innexin2 mutants and is recruited into ectopic expression domains defined by innexin2 misexpression. Conversely, RNA interference (RNAi) knockdown of innexin3 causes mislocalization of innexin2 and of DE-cadherin, causing cell polarity defects in the epidermis. Biochemical interaction studies, surface plasmon resonance analysis, transgenesis, and biochemical fractionation experiments demonstrate that both innexins interact via their C-terminal cytoplasmic domains during the assembly of heteromeric channels. Our data provide the first molecular and functional demonstration that innexin heteromerization occurs in vivo and reveal insight into a molecular mechanism by which innexins may oligomerize into heteromeric gap junction channels.

We recently demonstrated that heteromerization of innexins 2 and 3 from Drosophila melanogaster (Dm) is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins are thought to interact via their C-terminal cytoplasmic domains during the assembly of heteromeric gap junction channels. However, the mechanisms that control heteromeric versus homomeric channel formation are still largely unknown. Here we report the isolation of both non-modified and 2'-fluoro-2'-deoxy-modified RNA anti-innexin 2 aptamers by in vitro selection. The aptamers bind to a proximal epitope on the carboxyl-tail of Dm innexin 2 protein and specifically inhibit the heterologous interaction of innexin 2 and innexin 3 carboxyl-termini in vitro. These domain-specific inhibitors represent the first step towards functional studies focusing on the activity of these domains in vivo.