Engineered Cx26 variants established functional heterotypic Cx26/Cx43 and Cx26/Cx40 gap junction channels

Abstract

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.

A Cell Junctional Protein Network Associated with Connexin-26

“…CX26 association with other CX has also been disclosed by global interactome analyses [ 33 ]. CX26 physical interaction with paralogues is, therefore, a common feature since it is for other family members [ 43 ]. Few additional binding partners have been reported for CX26.…”

Concatenation of Human Connexin26 (hCx26) and Human Connexin46 (hCx46) for the Analysis of Heteromeric Gap Junction Hemichannels and Heterotypic Gap Junction Channels

“…For hCx46, the N188 formed corresponding hydrogen bonds with R180, T189, and D191. The importance of N176 and K168 for the docking interaction was recently demonstrated by Karademir et al (2016) [ 21 ]. The authors showed that by adapting the homologous residues, heterotypic docking between Cx26 and Cx40 connexons could be achieved.…”

A novel autosomal recessiveGJB2-associated disorder: Ichthyosis follicularis, bilateral severe sensorineural hearing loss, and punctate palmoplantar keratoderma

“…The gap junction channels formed by different connexins are functionally distinct with respect to their gating, conductance, and permeability characteristics. The complexity of these channels is emphasized by the fact that hemichannels and complete gap junction channels can be either homotypic or heterotypic (Karademir, Aoyama, Yue, Chen, & Bai, ; Koval, Molina, & Burt, ). Consequently, genetic mutations in a single connexin gene may impact formation of many types of channels, thus contributing to the phenotypic spectrum of diseases that result from such mutations.…”