Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions
Authors: Becker D. L. , Evans W. H. , Green C. R. , Warner Anne 
Published: April 2021

ABSTRACT
Gap junctions allow direct communication between cells without recourse to the extracellular space and have been widely implicated as important mediators of cell-cell signalling. They are constructed from the connexin proteins, which form a large family, and individual connexins show complex spatial and temporal variations in their expression patterns. Understanding how this variation contributes to the control of intercellular signalling, both in the adult and during embryonic development, is an important problem that would be aided by reagents that interfere with gap junctional communication through specific connexins. We have begun to address this issue by raising antibodies to peptides derived from connexin43 and connexin32. Connexin43 peptides were located in the amino terminus, cytoplasmic loop and carboxytail. Connexin32 peptides came from the cytoplasmic loop and the first extracellular loop. Immunoblotting and immunostaining properties of purified IgGs were characterized on mouse heart, liver and the 8- to 16-cell mouse embryo. Effects on transfer through gap junctions were assessed in the fully compacted 8-cell mouse embryo by co-injection with Lucifer Yellow or Cascade Blue. Embryos were maintained in culture to assess the developmental consequences of injection. Peptide competition was used to confirm the specificity of immunostaining and inhibition of dye transfer. All connexin specific antibodies recognized their parent connexin on immunoblots and showed no 43/32 cross-reactivity. The connexin32 extracellular loop antibody recognized both connexin 32 and 43 on immunoblots, as predicted by the amino acid sequence homology in this region, but did not immunostain intact gap junctions. Connexin specific anti-bodies that immuno-stained showed the predicted connexin specificity. Antibodies to either connexin43 amino acids (AA) 1-16 (amino terminus) or AA 101-112 (cytoplasmic loop) neither immunostained nor prevented functional communication through 8-cell embryo gap junctions. Anti-bodies to AA 123-136 and AA 131-142 in the cytoplasmic loop immunostained heart and 8-cell embryo gap junctions and blocked transfer through them with high efficiency. Fab′ fragments were equally effective. Peptide competition showed that both antibodies contained epitopes within AA 131-136 of connexin43. Antibodies against AA 313-324 in the carboxytail immunostained heart and the 8-cell embryo and, as IgGs, prevented dye transfer. Fab′ fragments were ineffective. All connexin43 antibodies that blocked gap junctional communication between cells of the 8-cell mouse embryo induced non-communicating cells subsequently to withdraw from compaction. Connexin32 cytoplasmic loop antibodies stained mouse liver, but did not stain 8-cell mouse embryo gap junctions, did not influence transfer through them and had no influence on subsequent development. Connexin26 antibodies also did not stain 8-16 cell mouse embryos. Our results suggest that for connexin43, the cytoplasmic loop region encompassing AA 131-136 is important for transfer through connexin43 gap junctions and eliminate AA 313-324 from functional control. They support the view that connexin43 is the predominant connexin protein expressed at the 8-cell stage in the mouse embryo and show that communication through gap junctions containing connexin43 is essential for the maintenance of compaction.