Most of the early studies on gap junction (GJ) channel function and docking compatibility were on rodent connexins, while recent research on GJ channels gradually shifted from rodent to human connexins largely due to the fact that mutations in many human connexin genes are found to associate with inherited human diseases. The studies on human connexins have revealed some key differences from those found in rodents, calling for a comprehensive characterization of human GJ channels. Functional studies revealed that docking and formation of functional GJ channels between two hemichannels are possible only between docking-compatible connexins. Two groups of docking-compatible rodent connexins have been identified. Compatibility is believed to be due to their amino acid residue differences at the extracellular loop domains (E1 and E2). Sequence alignment of the E1 and E2 domains of all connexins known to make GJs revealed that they are highly conserved and show high sequence identity with human Cx26, which is the only connexin with near atomic resolution GJ structure. We hypothesize that different connexins have a similar structure as that of Cx26 at the E1 and E2 domains and use the corresponding residues in their E1 and E2 domains for docking. Based on the Cx26 GJ structure and sequence analysis of well-studied connexins, we propose that the E1-E1 docking interactions are staggered with each E1 interacting with two E1s on the docked connexon. The putative E1 docking residues are conserved in both docking-compatible and -incompatible connexins, indicating that E1 does not likely serve a role in docking compatibility. However, in the case of E2-E2 docking interactions, the putative docking residues are only conserved within the docking-compatible connexins, suggesting the E2 is likely to serve the function of docking compatibility. Docking compatibility studies on human connexins have attracted a lot of attention due to the fact that putative docking residues are mutational hotspots for several connexin-linked human diseases. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Gap junctions formed by different connexins are expressed throughout the body and harbour unique channel properties that have not been fully defined mechanistically.r Recent structural studies by cryo-electron microscopy have produced high-resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure-function comparison.r Here, we conducted comparative molecular dynamics simulation and electrophysiology studies to dissect the isoform-specific differences in Cx46 and Cx50 intercellular channel function.r We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N-terminal domains, in particular the residue at the 9th position and differences in hydrophobic anchoring sites.r The results of this study establish the open state Cx46/50 structural models as archetypes for structure-function studies targeted at elucidating the mechanism of gap junction channels and the molecular basis of disease-causing variants.

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Gap junction (GJ) channels mediate direct intercellular communication and are composed of two docked hemichannels (connexin oligomers). It is well documented that the docking and formation of GJs are possible only between compatible hemichannels (or connexins). The mechanisms of heterotypic docking compatibility are not fully clear. We aligned the protein sequences of docking-compatible and -incompatible connexins with that of connexin26 (Cx26). We found that two docking hydrogen bond (HB)-forming residues on the second extracellular domain (E2) of Cx26 and their equivalent residues are well conserved within docking-compatible connexins, but different between docking-incompatible connexins. Replacing one or both of these residues of Cx26 into the corresponding residues in the docking incompatible connexins (K168V, N176H or K168V-N176H) increased the formation of morphological and functional heterotypic GJs with connexin43 (Cx43) or connexin40 (Cx40), indicating that these two residues are important for docking incompatibility between Cx26 and these connexins. Our homology structure models predict that both HBs and hydrophobic interactions at the E2 docking interface are important docking mechanisms in heterotypic Cx26 K168V-N176H/Cx43 GJs and probably other docking compatible connexins. Revealing the key residues and mechanisms of heterotypic docking compatibility will assist us in understanding why these putative docking residues are hotspots of disease-linked mutants.

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