Figure 7.

Cx43-mediated coupling is disturbed in ODDD-mutated mice and can be maintained by Cx43/Cx43 homotypic and Cx43G138R-Cx43WT heterotypic channels. Intercellular transfer of microinjected neurobiotin in wild-type Cx43-expressing cells [primary mouse cardiomyocytes (A and C), ES cells (E) and HeLa cells (G)] in comparison with Cx43G138R-expressing cells [primary mouse cardiomyocytes (B and D), ES cells (F) and HeLa cells (H)]. In (C) and (D), the coupling area is outlined in pseudo-color. The diagrams shown in (I) compare neurobiotin transfer in wild-type and mutated cardiomyocytes and stem cells as well as in HeLa cells expressing Cx43WT or Cx43G138R alone or co-expressing Cx43WT and Cx43G138R. A residual coupling in approximately 2.5 cells in contact (~6.4%) was observed. In stem cells dye, transfer was substantially higher (six cells; 20.8%). HeLa cells expressing Cx43G138R alone or in parallel with Cx43WT showed a very low level of dye transfer. Cx43–eGFP/Cx43G138R heterotypic junctions, shown in (J), form junctional plaques (see arrows) in the junctional region between HeLa Cx43G138R cell (left/top) exhibiting homogenous eGFP in cytoplasm and HeLaCx43-eGFP cell (right/bottom) exhibiting punctate staining of eGFP prominently in contacting membranes. (K) Summarized junctional conductance dependence on transjunctional voltage (G_j–V_j) measured in nine cell pairs forming heterotypic Cx43–eGFP/Cx43G138R junctions. These exhibit an asymmetric V_j gating with higher sensitivity at positive voltages on the HeLaCx43-eGFP side. (L) Unitary opening and closing events at the single-channel level of Cx43–eGFP/Cx43G138R heterotypic GJ channels seen as stepwise junctional current (I_j) transitions in response to consecutive negative and positive voltage steps applied to the HeLaCx43-eGFP cell. Channels show a tendency to open during a negative voltage step and to close during a positive voltage step that is in accordance with macroscopic G_j–V_j dependence shown in (K). (M) Single-channel record demonstrating that both fast and slow gating mechanisms operate in Cx43G138R/Cx43 heterotypic GJC. During application of hyperpolarizing V_j step to HeLa Cx43G138R cell, the channel operates mainly between the open state with a conductance of ~103 pSand the residual state with a conductance of ~25 pS. Arrows point totransitions between the open state and the fully closed state, which can be ascribed to the operation of slow gating mechanism.