Gap junctions result from the docking of two hemichannels.Homomeric heterotypic gap junctions can be formed only between compatible connexins.Previous studies indicated that the extracellular loop2 (E2) is an important molecular domain for heterotypic docking selectivity.The detailed residues in E2 responsible for docking compatibility remain elusive.Based on the crystal structure of Cx26 gap junction channel at 3.5(A) resolution, we developed homology models of heterotypic channels and analyzed docking selectivity with various hemichannel pairs.We found that the hydrogen bonds between E2 domains are conserved in several heterotypically compatible hemichannels, e.g.between Cx26 and Cx32.According to our model analysis, a total of 60 hydrogen bonds (36 of them at E2-E2 interfaces) could be indentified between a pair of Cx32 and Cx26 hemichannels.Asparagine 175 in E2 domain of Cx32 was a pivotal residue forming 3 hydrogen bonds with K168, T177 and D179 of interdocked Cx26.Cx32 mutations, N175Y or N175H, destroy 3/3 or 2/3 hydrogen bonds at E2-E2 docking interface, respectively, at one pair Cx26/Cx32 heterotypic docking interface.Our model predicts that the Cx32 N 175Y and N 175H hemichannels are unlikely to dock with Cx26 hemichannels properly due to steric hindrance and the substantial loss of hydrogen bonds at the docking interfaces.