title: A mechanism of gap junction docking revealed by functional rescue of a human-disease-linked connexin mutant.
abstract: Gap junctions are unique intercellular channels formed by the proper docking of two hemichannels from adjacent cells. Each hemichannel is a hexamer of connexins (Cxs) - the gap junction subunits, which are encoded by 21 homologous genes in the human genome. The docking of two hemichannels to form a functional gap junction channel is only possible between compatible Cxs, but the underlying molecular mechanism is unclear. On the basis of the crystal structure of the Cx26 gap junction, we developed homology models for homotypic and heterotypic channels from Cx32 and/or Cx26; these models predict six hydrogen bonds at the docking interface of each pair of the second extracellular domain (E2). A Cx32 mutation N175H and a human-disease-linked mutant N175D were predicted to lose the majority of the hydrogen bonds at the E2 docking-interface; experimentally both mutations failed to form morphological and functional gap junctions. To restore the lost hydrogen bonds, two complementary Cx26 mutants - K168V and K168A were designed to pair with the Cx32 mutants. When docked with Cx26K168V or K168A, the Cx32N175H mutant was successfully rescued morphologically and functionally in forming gap junction channels, but not Cx32 mutant N175Y. By testing more homotypic and heterotypic Cx32 and/or Cx26 mutant combinations, it is revealed that a minimum of four hydrogen bonds at each E2-docking interface are required for proper docking and functional channel formation between Cx26 and Cx32 hemichannels. Interestingly, the disease-linked Cx32N175D could be rescued by Cx26D179N, which restored five hydrogen bonds at the E2-docking interface. Our findings not only provide a mechanism for gap junction docking for Cx26 and Cx32 hemichannels, but also a potential therapeutic strategy for gap junction channelopathies.
authors.affiliation: Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada N6A 5C1.
authors.forename: Xiang-Qun
authors.initials: XQ
authors.lastname: Gong
authors.forename: So
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Tomitake
authors.initials: T
authors.lastname: Tsukihara
authors.forename: Donglin
authors.initials: D
authors.lastname: Bai
mesh_terms: D000818:Animals; D053447:Channelopathies / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D007473:Ion Channels / Q000378:metabolism*; D051379:Mice; D008956:Models, Chemical; D058990:Molecular Targeted Therapy; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D055503:Protein Multimerization / Q000235:genetics; D016415:Sequence Alignment; D012984:Software; D000097002:Gap Junction beta-1 Protein
chemical_list: D000072259:Connexin 26; D017630:Connexins; D007473:Ion Channels; D000097002:Gap Junction beta-1 Protein; C000607042:GJB2 protein, human
keywords: Docking; Dual whole cell patch-clamp; Gap junction channel; Homology modelling; Hydrogen bond