T. Toyofuku et al., DIRECT ASSOCIATION OF THE GAP JUNCTION PROTEIN CONNEXIN-43 WITH ZO-1 IN CARDIAC MYOCYTES, The Journal of biological chemistry, 273(21), 1998, pp. 12725-12731
The gap junction protein connexin-43 is normally located at the interc
alated 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, localizat
ion of connexin 43 likely involves an interaction with the cytoskeleto
n; immunofluorescence microscopy showed that in cardiac myocytes, conn
exin-43 specifically colocalizes with the cytoskeletal proteins ZO-1 a
nd cu-spectrin. In transfected HEK293 cells, immunoprecipitation exper
iments using coexpressed epitope-tagged connexin-43 and ZO-1 indicated
that ZO-1 links connexin-43 with alpha-spectrin, The domains responsi
ble for the protein-protein interaction between connexin-43 and ZO-1 w
ere identified using affinity binding assays with deleted ZO-1 and con
nexin-43 fusion proteins, Immunoblot analysis of associated proteins s
howed that the C-terminal domain of connexin-43 binds to the N-termina
l domain of ZO-1, The role of this linkage in gap junction formation w
as examined by a dominant-negative assay using the N-terminal domain o
f 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 redistri
bution of connexin-43 coincided with a loss of electrical coupling. We
therefore conclude that the linkage between connexin-43 and alpha-spe
ctrin, via ZO-1, may serve to localize connexin-43 at the intercalated
discs, thereby generating functional gap junctions in cardiac myocyte
s.