Structural and molecular mechanisms of gap junction remodeling in epicardial border zone myocytes following myocardial infarction.

Lateralization of the ventricular gap junction protein connexin 43 (Cx43) occurs in epicardial border zone myocytes following myocardial infarction (MI) and is arrhythmogenic. Alterations in Cx43 protein partners have been hypothesized to play a role in lateralization although mechanisms by which this occurs are unknown. To examine potential mechanisms we did nuclear magnetic resonance, yeast 2-hybrid, and surface plasmon resonance studies and found that the SH3 domain of the tyrosine kinase c-Src binds to the Cx43 scaffolding protein zonula occludens-1 (ZO-1) with a higher affinity than does Cx43. This suggests c-Src outcompetes Cx43 for binding to ZO-1, thus acting as a chaperone for ZO-1 and causing unhooking from Cx43. To determine whether c-Src/ZO-1 interactions affect Cx43 lateralization within the epicardial border zone, we performed Western blot, immunoprecipitation, and immunolocalization for active c-Src (p-cSrc) post-MI using a canine model of coronary occlusion. We found that post-MI p-cSrc interacts with ZO-1 as Cx43 begins to decrease its interaction with ZO-1 and undergo initial loss of intercalated disk localization. This indicates that the molecular mechanisms by which Cx43 is lost from the intercalated disk following MI includes an interaction of p-cSrc with ZO-1 and subsequent loss of scaffolding of Cx43 leaving Cx43 free to diffuse in myocyte membranes from areas of high Cx43, as at the intercalated disk, to regions of lower Cx43 content, the lateral myocyte membrane. Therefore shifts in Cx43 protein partners may underlie, in part, arrhythmogenesis in the post-MI heart.

Characterization of the pH-dependent interaction between the gap junction protein connexin43 carboxyl terminus and cytoplasmic loop domains.

A prevailing view regarding the regulation of connexin43 (Cx43) gap junction channels is that, upon intracellular acidification, the carboxyl-terminal domain (Cx43CT) moves toward the channel opening to interact with specific residues acting as a receptor site. Previous studies have demonstrated a direct, pH-dependent interaction between the Cx43CT and a Cx43 cytoplasmic loop (Cx43CL) peptide. This interaction was dependent on alpha-helical formation for the peptide in response to acidification; more recent studies have shown that acidification also induces Cx43CT dimerization. Whether Cx43CT dimerization is an important structural component in Cx43 regulation remains to be determined. Here we used an assortment of complimentary biophysical techniques to characterize the binding of Cx43CT or its mutants to itself and/or to a more native-like Cx43CL construct (Cx43CL(100-155), residues 100-155). Our studies expand the observation that specific Cx43CT domains are important for dimerization. We further show that properties of the Cx43CL(100-155) are different from those of the Cx43CL peptide; solvent acidification leads to Cx43CL(100-155) oligomerization and a change in the stoichiometry and binding affinity for the Cx43CT. Homo-Cx43CT and Cx43CL(100-155) oligomerization as well as the Cx43CT/Cx43CL(100-155) interaction can occur under in vivo conditions; moreover, we show that Cx43CL(100-155) strongly affects resonance peaks corresponding to Cx43CT residues Arg-376-Asp-379 and Asn-343-Lys-346. Overall, our data indicate that many of the sites involved in Cx43CT dimerization are also involved in the Cx43CT/Cx43CL interaction; we further propose that chemically induced Cx43CT and Cx43CL oligomerization is important for the interaction between these cytoplasmic domains, which leads to chemically induced gating of Cx43 channels.

The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog 1.

Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.