Hydrogen Bonds at the Docking Interface are Critical for Functional Gap Junction Channel Formation of Cx26 and Cx32

Abstract

Gap junctions are unique intercellular channels formed by proper docking of two hemichannels from adjacent cells; each hemichannel is a hexamer of connexins - the gap junction proteins encoded by 21 homologous genes in human genome. Docking of two hemichannels to form a functional gap junction channel is only possible between the hemichannels formed by compatible connexins. The underlying docking mechanism for compatibility is not clear. Based on the crystal structure of Cx26 gap junction channel, we developed homology structural models and generated a series of mutants on extracellular domain 2 (E2). According to the model, 36 hydrogen bonds (HBs) were indentified at E2-E2 interface between a pair of Cx32/Cx26 hemichannels. The HB-forming residues between E2 domains are conserved in Cx32, Cx26 and heterotypically compatible connexins, but not in non-compatible connexins. Asp175 of Cx32 was a pivotal residue forming 3 HBs with K168, T177 and D179 of inter-docked Cx26. Cx32 mutations, N175Y or N175H, destroy 3/3 or 2/3 HBs, respectively, at the E2-E2 docking interface. Experimentally, these two mutants failed to form putative gap junction plaques and were unable to form homotypic functional gap junction channels. Morphological and functional tests of various combinations of these mutants with designed mutant and wild-type Cx26 revealed that the hydrogen bonds at the E2 docking interface are critical for docking compatibility in the gap junctions formed by Cx26 and/or Cx32. Restoring more hydrogen bonds at the docking interface was able to rescue the function of Cx32N175H, but not N175Y using designed mutant or wild-type Cx26. Our results demonstrate that HBs at the E2-E2 docking interface are key factors for heterotypic docking compatibility between Cx32 and Cx26 hemichannels and possibly other hemichannels formed by compatible connexins.