A gap junction docking mechanism revealed by functional rescue of a human disease-linked connexin mutant

Abstract

SummaryGap 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.

Data of the molecular dynamics simulations of mutations in the human connexin46 docking interface

“…Despite the reported rescue function of such reciprocal charge mutations for the Cx32/Cx26 complex [4], [9], the Cx46 complex dissociated within 50 ns in our simulations.…”

“…The reciprocal charge mutations N188D and D191N on Cx46 did not stabilize the complex, despite the reported rescue function of such reciprocal charge mutations for the Cx32/Cx26 complex (Gong et al [9]), the Cx46 complex dissociated within 50 ns simulations time in our model.…”

“…According to Gong et al [9], the stability of a gap junction channel is achieved if an average of 18 HBs is achieved. For the hCx46N188Q channel the number of HBs is reduced to less than 10 within the first 3 ns leading to the dissociation of the complex.…”

Gap junction structure: unraveled, but not fully revealed

“…The mutated amino acid in Cx46 (N188) corresponds to N176 of Cx26. A mutant of Cx32 at the corresponding position (Cx32N175D) causes X-linked Charcot-Marie-Tooth disease and, similarly, does not form functional homotypic gap junction channels 51 .…”

“…Amino acids in EL2 have been implicated in determining connexin compatibility for the formation of functional heterotypic channels, based on the results of expression studies of chimeric connexins in which the EL1 or EL2 domains were replaced with those from a different, incompatible connexin 53, 54 . Homology modeling and studies of the ability of Cx32 mutants to form heterotypic channels with Cx26 suggest that at least four hydrogen bonds between a pair of opposed EL2 domains are required for proper docking and functional heterotypic channel formation 51 .…”