c-Src regulates the interaction between connexin-43 and ZO-1 in cardiac myocytes

Connexin-43 is known to interact directly with ZO-1 in cardiac myocytes, but little is known about the role of ZO-1 in connexin-43 function. In cardiac myocytes, constitutively active c-Src inhibited endogenous interaction between connexin-43 and ZO-1 by binding to connexin-43. In HEK293 cells, by contrast, a connexin-43 mutant lacking the Src phosphorylation site (Tyr265) interacted with ZO-1 despite cotransfection of a constitutively active c-Src. Moreover, in vitro binding assays using recombinant proteins synthesized from regions of connexin-43 and ZO-1 showed that the tyrosine-phosphorylated C terminus of connexin-43 interacts with the c-Src SH2 domain in parallel with the loss of its interaction with ZO-1. Cell surface biotinylation revealed that, by phosphorylating Tyr265, constitutively active c-Src reduces total and cell surface connexin-43 down to the levels seen in cells expressing a mutant connexin-43 lacking the ZO-1 binding domain. Finally, electrophysiological analysis showed that both the tyrosine phosphorylation site and the ZO-1-binding domain of connexin-43 were involved in the regulation of gap junctional function. We therefore conclude that c-Src regulates the interaction between connexin-43 and ZO-1 through tyrosine phosphorylation and through the binding of its SH2 domain to connexin-43.