In situ structure of a gap junction - stomatin complex

Gap junctions (GJ) are intercellular channels that mediate electrical signals and the transfer of small molecules. GJs are crucial for the functions of the brain, heart and other organs. While structures of purified homomeric GJs are available, we lack in situ structures. In vivo, GJs can form heteromers with different functionalities, and may associate with other proteins. Here, we analyzed Caenorhabditis elegans GJs by cryo-electron tomography and sub-tomogram averaging. We observed hexagonal arrays of GJs at cellular junctions in primary embryonal cell culture that displayed distinct wide and narrow conformations. Moreover, in about 20% of the observed channels, we found a cap-like, cytosolic protein assembly enclosing the channel pore. We propose that the cap-structure is formed by the stomatin UNC-1, which is known to interact with C. elegans GJs, and strengthen this hypothesis by matching AlphaFold3 models of UNC-1 multimers with our GJ average. Furthermore, expressing UNC-1 and the C. elegans innexin UNC-9 in HEK cells resulted in similar structures at cell-cell contacts. UNC-1/stomatin ring assemblies may affect GJ formation or functions like rectification, that might be evolutionarily conserved. Significance StatementGap junction (GJ) channels connect neighboring cells. Structures of (purified) GJs have been studied in vitro, but not in situ. We identified GJ channels in primary Caenorhabditis elegans cells by cryo-electron tomography, and analyzed their structure by sub-tomogram averaging. The channels transverse the membranes of connected cells, and AlphaFold3 (AF3) models of the GJ subunit UNC-9, assuming dodecamers, fit the experimentally obtained surface map well. We observed a cytosolic cap structure on the GJ channels. The stomatin protein UNC-1 is known to physically interact with UNC-9 GJs. AF3 models of UNC-1 hexadecamers fit the cap structure, indicating that it may be formed by UNC-1, providing a first idea how UNC-1 interacts with, and may functionally influence, GJ channels.