Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule

Connexin molecules form intercellular membrane channels facilitating electronic coupling and the passage of small molecules between adjoining cells. Connexin26 (Cx26) is the second smallest member of the gap junction protein family, and mutations in Cx26 cause certain hereditary human diseases such as skin disorders and hearing loss. Here, we report the electron crystallographic structure of a human Cx26 mutant (M34A). Although crystallization trials used hemichannel preparations, the density map revealed that two hemichannels redocked at their extracellular surfaces into full intercellular channels. These orthorhombic crystals contained two sets of symmetry-related intercellular channels within three lipid bilayers. The 3D map shows a prominent density in the pore of each hemichannel. This density contacts the innermost helices of the surrounding connexin subunits at the bottom of the vestibule. The density map suggests that physical blocking may play an important role that underlies gap junction channel regulation. Our structure allows us to suggest that the two docked hemichannels can be independent and may regulate their activity autonomously with a plug in the vestibule.