Gating properties of gap junction channels assembled from connexin43 and connexin43 fused with green fluorescent protein

We used cell lines expressing wild-type connexin43 (Cx43) and Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gap junction channel gating. Previously it was suggested that each hemichannel in a cell-cell channel possesses two gates, a fast gate that closes channels to a nonzero conductance or residual state via fast (< similar to2 ms) transitions and a slow gate that fully closes channels via slow transitions (> similar to 10 ms). Here we demonstrate that transjunctional voltage (V-j) regulates both gates and that they are operating in series and in a contingent manner in which the state of one gate affects gating of the other. Cx43-EGFP channels lack fast V-j gating to a residual state but show slow V-j gating. Both Cx43 and Cx43-EGFP channels exhibit slow gating by chemical uncouplers such as CO2 and alkanols. Chemical uncouplers do not induce obvious changes in Cx43-EGFP junctional plaques, indicating that uncoupling is not caused by dispersion or internalization of junctional plaques. Similarity of gating transitions during chemical gating and slow V-j gating suggests that both gating mechanisms share common structural elements. Cx43/Cx43-EGFP heterotypic channels showed asymmetrical V-j gating with fast transitions between open and residual states only when the Cx43 side was relatively negative. This result indicates that the fast V-j gate of Cx43 hemichannels closes for relative negativity at its cytoplasmic end.