Direct Association of the Gap Junction Protein Connexin-43 with ZO-1 in Cardiac Myocytes

• Published in: The Journal of biological chemistry Vol. 273; no. 21; pp. 12725 - 12731
• Main Authors: Toyofuku, Toshihiko, Yabuki, Masanori, Otsu, Kinya, Kuzuya, Tsunehiko, Hori, Masatsugu, Tada, Michihiko
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.05.1998; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Cell Line; Connexin 43 - metabolism; DNA, Complementary; Humans; Membrane Proteins - metabolism; Mutagenesis; Myocardium - cytology; Myocardium - metabolism; Phosphoproteins - metabolism; Rats; Rats, Wistar; Spectrin - metabolism; Zonula Occludens-1 Protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.21.12725

The gap junction protein connexin-43 is normally located at the intercalated discs of cardiac myocytes, and it plays a critical role in the synchronization of their contraction. The mechanism by which connexin-43 is localized within cardiac myocytes is unknown. However, localization of connexin-43 likely involves an interaction with the cytoskeleton; immunofluorescence microscopy showed that in cardiac myocytes, connexin-43 specifically colocalizes with the cytoskeletal proteins ZO-1 and α-spectrin. In transfected HEK293 cells, immunoprecipitation experiments using coexpressed epitope-tagged connexin-43 and ZO-1 indicated that ZO-1 links connexin-43 with α-spectrin. The domains responsible for the protein-protein interaction between connexin-43 and ZO-1 were identified using affinity binding assays with deleted ZO-1 and connexin-43 fusion proteins. Immunoblot analysis of associated proteins showed that the C-terminal domain of connexin-43 binds to the N-terminal domain of ZO-1. The role of this linkage in gap junction formation was examined by a dominant-negative assay using the N-terminal domain of ZO-1. Overexpression of the N-terminal domain of ZO-1 in connexin-43-expressing cells resulted in redistribution of connexin-43 from cell-cell interfaces to cytoplasmic structures; this intracellular redistribution of connexin-43 coincided with a loss of electrical coupling. We therefore conclude that the linkage between connexin-43 and α-spectrin, via ZO-1, may serve to localize connexin-43 at the intercalated discs, thereby generating functional gap junctions in cardiac myocytes.