Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes.
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 alpha-spectrin. In transfected HEK293 cells, immunoprecipitation experiments using coexpressed epitope-tagged connexin-43 and ZO-1 indicated that ZO-1 links connexin-43 with alpha-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 alpha-spectrin, via ZO-1, may serve to localize connexin-43 at the intercalated discs, thereby generating functional gap junctions in cardiac myocytes.
Toyofuku
T
T
Department of Medicine and Pathophysiology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan. toyofuku@mr-path.med.osaka-u.ac.jp
Yabuki
M
M
Otsu
K
K
Kuzuya
T
T
Hori
M
M
Tada
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D018031:Connexin 43 / Q000378:metabolism*; D018076:DNA, Complementary; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D016296:Mutagenesis; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013049:Spectrin / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
D018031:Connexin 43; D018076:DNA, Complementary; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D013049:Spectrin