Deep evolutionary origins of the connexin gene family

The vertebrate connexin family encodes a diverse repertoire of transmembrane channels that mediate both intercellular communication and cytosol to extracellular space exchange. Their apparent absence in non-chordates has led to the prevailing view that connexins arose during chordate evolution with analogous functions performed in non-chordates by the innexins, a structurally similar but non-homologous protein family. Here, using a combination of sequence and structural analyses, we discover the presence of connexins in anthozoan and protostome genomes despite widespread secondary gene loss in Bilateria. We show that these connexins can be present in up to 100 copies per genome and are often found as N-terminal domains of NOD-like receptors (NLRs) or other immune genes. We show that these invertebrate connexins localise at the cell membrane, function as either hemichannels (anthozoans and protostomes) or gap junctions (protostomes), and respond to depolarisation enabling the transfer of small molecules. Further, we demonstrate that a subset of the invertebrate connexin-like domains are sensitive to CO ₂ and mirroring findings in vertebrate connexins, the CO ₂ -mediated gating stems from the formation of a carbamate bridge between neighbouring subunits. Taken together, we trace the evolution of this gene family to the cnidarian-bilaterian ancestor, demonstrate functional similarity between invertebrate and vertebrate connexins, and suggest an ancient role of connexins in innate immunity.