Heterotypic connexin50/connexin50 mutant gap junction channels reveal interactions between two hemichannels during transjunctional voltage-dependent gating.

Key points Gap junctions (GJs) composed of two hemichannels are intercellular channels that provide a direct pathway for the exchange of ions and signalling molecules between adjoining cells., GJ conductance is regulated by transjunctional voltage ( Vj, the voltage difference between the interiors of adjoining cells), known as Vj-dependent gating., The GJs made up of two identical hemichannels show mirror symmetrical Vj-dependent gating to positive and negative Vj polarities; GJs formed by two non-identical hemichannels show novel Vj-gating properties that cannot be simply ascribed to the predicted functional properties of the component hemichannels., We provide experimental evidence at both macroscopic and single channel levels that two hemichannels interact with each other in a complex manner for the Vj-dependent gating phenotype of a GJ., The results help reveal the molecular mechanisms of the gating properties of GJs formed by two non-identical hemichannels., Abstract To investigate transjunctional voltage ( Vj)-dependent gating mechanisms of connexin50 (Cx50) gap junction (GJ) channels and to elucidate the relative contribution of each hemichannel of a heterotypic GJ channel to Vj-dependent gating, we performed dual voltage-clamp recordings on heterotypic GJ channels formed by Cx50 and a mutant, Cx50N9R or a chimera, Cx50-Cx36N. Our results provide evidence that the two component hemichannels interact with each other during Vj-dependent gating. Cx50/Cx50N9R heterotypic GJ channels exhibited asymmetrical Vj-dependent gating which cannot be ascribed to the function of an individual hemichannel for a certain polarity of voltage; instead it can only be ascribed to the combined effects of both hemichannels. Single GJ channel open dwell-time analyses showed that homotypic Cx50 channels adopted short-lived and long-lived open states. Heterotypic combinations of Cx50/Cx50N9R gave rise to shorter mean dwell-times when Cx50-expressing cells received relatively positive Vj, and longer mean dwell-times when positive Vj was applied at the Cx50N9R side. In contrast, Cx50/Cx50-Cx36N heterotypic channels showed asymmetrical Vj-dependent gating, which appears to be caused by enhanced and reduced Vj-gating sensitivity of Cx50-Cx36N and Cx50 hemichannels, respectively. Unitary conductance of the main open state of both types of heterotypic GJ channel cannot be simply predicted by assuming a Vj redistribution across the two hemichannels arranged in series in heterotypic GJ channels. Our data also reveal reasons for the invisibility of fast Vj-gating transitions from open to substate in homotypic Cx50N9R and Cx50-Cx36N channels. [ABSTRACT FROM AUTHOR]