Abstract-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.
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, pHdependent interaction between the Cx43CT and a Cx43 cytoplasmic loop (Cx43CL) peptide. This interaction was dependent on ␣-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.Gap junctions are integral membrane proteins that enable direct cytoplasmic exchange of ions and low molecular weight metabolites between adjacent cells. They provide a pathway for the propagation and/or amplification of signal transduction cascades triggered by cytokines, growth factors, and other cell signaling molecules involved in growth regulation and development. Mutations in the gap junction proteins have been associated with hereditary non-syndromic deafness, Charcot-MarieTooth disease, oculodentodigital dysplasia, and cataracts, among other congenital human diseases (1). Gap junctions are formed by the apposition of connexons from adjacent cells, where each connexon is formed by six connexin proteins. Connexins are tetraspan transmembrane domain proteins with intracellular amino and carboxyl termini. There are 21 different connexin genes in the human genome. Although there is significant sequence homology among connexins, the major divergence in primary structures occurs in the cytoplasmic domains. The 43-kDa protein connexin43 (Cx43) 2 is the most abundant gap junction protein in various tissues (e.g. heart and brain) and is the focus of the present study.The importance of Cx43 gap junc...
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.Connexins, of which there are more than 20 isoforms in mice and humans, are tetraspan proteins that oligomerize to form hexameric connexons. Connexons contributed by each cell interact head-to-head across the extracellular space, forming channels providing direct cytoplasmic continuity between cells.Aggregates of these channels, gap junctions, are found in almost all tissues. Connexins have intracellular amino-terminal cytoplasmic loops and carboxyl-terminal domains. Although much of the connexin sequence is highly conserved throughout the protein family, the amino acid sequences of intracellularly localized cytoplasmic loop and carboxyl-terminal domains vary markedly between connexin isoforms, and these regions likely confer isoform specificity. Carboxyl-terminal connexin domains, in particular, contain multiple sites for protein-protein interactions. For example, the major gap junction protein of heart and astrocytes, Connexin43 (Cx43), 2 possesses binding sites for Src homology 3 (SH3), SH2, WW, MAPK (mitogenactivated protein kinase), and PDZ within its carboxyl-terminal domain, and other connexins, although less well mapped, also display potential binding sites. The function of these proteinprotein interactions is not well understood, but many of them regulate the function of the gap junction channel (1). Interestingly, analysis of the primary sequence of the ␣ subdivision of connexins (which includes Cx43) for consensus binding sites indicates that many of these connexins have potential PDZ in...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.