Real-time and simulataneous analysis of gap junctional structure and function in living cells

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1. Real-time visualization of gap junctions in living cells using connexin 43-GFP fusion proteinWe used connexin 43-green fluorescent protein (Cx43-GFP) fusion protein in combination with confocal laser scanning microscopy. The rat Cx43 gene was fused to the gene encoding GFP and transfected into human HeLa cells to generate stable lines constitutively expressing Cx43-GFP. Permanent cell lines expressing Cx43-GFP fusion protein and only GFP were obtained by culture in selection medium containing G418. Cx43-GFP fusion protein was localized as spots, plaques or lines mainly on plasma membranes between cells in contact, whereas GFP without Cx43 was expressed diffusely in the cytoplasm and nucleus. An assay of gap junctional intercellular communication using ethidium bromide dye microinjection transfer revealed that Cx43-GFP fusion protein formed functional gap junctions. Time-lapse microscopy after laser photobleaching showed that Cx43-GFP fusion protein dynamically moved not only on plas … More ma membranes but also in cytoplasm.2. Real-time visualization of gap junction distribution and cellular functionWe used Cx43-GFP fusion protein and a fluorescent Ca2+ indicator (Fura Red). Cx43-EGFP vector was transfected into cultured neonatal rat cardiomyocytes by lipofection. Simultaneous visualization of Cx43-EGFP and intracellular Ca2+ demonstrated that Ca2+ transients were synchronized between Cx43-EGFP-expressing myocytes and non-expressing myocytes, indicating that Cx43-EGFP did not inhibit gap junctional communication mediated by endogenous Cx43 expressed in the cardiomyocytes. The simultaneous visualization of connexin-GFP and cellular function provides a useful system for studies of gap junctions.3. Inhibition of gap junctional intercellular communication by use of dominant-negative Cx43-GFP expression vectorWe made a dominant-negative Cx43-green fluorescent protein (GFP) expression vector by sitedirected mutagenesis. When this vector was transfected into a liver epithelial cell line (LAR20) expressing wild-type Cx43, gap junctional intercellular communication via wild-type Cx43 was inhibited in dominant negative manners. This vector provides a useful system for studies of gap junctions. Less