Heterologous interactions between connexins of gap junctions

Gap junctions are intercellular channels formed by docking of two hexameric connexons. The structural units in vertebrates are connexins, which constitute a multigene family with at least 17 members identified from rodents. Many connexins are co-expressed in the same cells. Extensive studies of homomeric junctions have shown that each connexin imparts its unique properties to the gap junction channels. Thus it is of particular interest to investigate the properties of heterologous gap junction channels and the mechanisms for specificity of heterologous interactions.; Two forms of heterologous interactions between connexins are possible: heterotypic interactions happen between adjacent cells expressing different connexins, whereas heteromeric interactions occur between different connexin subunits in a connexon. Functional heterotypic interactions tend to occur within, but not between, two major connexin subfamilies (α and β), as defined by sequence homology. By substituting two residues in the second extracellular loop of Cx30.3 or Cx32, their docking specificity could be inverted (Chapter II). For example, heterotypic pairing preference of Cx32, typical β connexin, was switched to an α pattern in the mutated Cx32 (hence renamed as Cx32α). Therefore, these two residues represent a simple mechanism for docking specificity between α and β connexins. Further biochemical studies demonstrated that Cx32α and Cx26 could still form intact heterotypic junctional structures, although no channel activity was observed. This is the first evidence to discriminate physical docking from functional opening of gap junction channels.; The mechanism for docking specificity within a connexin subfamily was more complicated. Domain-exchange studies based on Cx40 and Cx43 (Chapter III) suggested that tertiary structures of extracellular loops, as influenced by the transmembrane domains or cytoplasmic domains, also exert influence on docking specificity.; Cx32α was used to further study the heteromeric interactions (Chapter IV). The fact that Cx32α no longer heterotypically interacts with Cx26 significantly simplifies the studies on properties and composition of heteromeric Cx32α/Cx26 channels. The stoichiometry of heteromeric channels is biased toward Cx26. In these channels, Cx26 dominates the size selectivity, while both connexins contribute to the charge selectivity. Strikingly, Cx32α dominates the voltage gating polarity although it is in the minority in heteromeric forms.