Regulation of cell-cell communication by the gap junction protein connexin43 can be modulated by a variety of connexin-associating proteins. In particular, c-Src can disrupt the connexin43 (Cx43)-zonula occludens-1 (ZO-1) interaction, leading to down-regulation of gap junction intercellular communication. The binding sites for ZO-1 and c-Src correspond to widely separated Cx43 domains (ϳ100 residues apart); however, little is known about the structural modifications that may allow information to be transferred over this distance. Here, we have characterized the structure of the connexin43 carboxyl-terminal domain (Cx43CT) to assess its ability to interact with domains from ZO-1 and c-Src. NMR data indicate that the Cx43CT exists primarily as an elongated random coil, with two regions of ␣-helical structure. NMR titration experiments determined that the ZO-1 PDZ-2 domain affected the last 19 Cx43CT residues, a region larger than that reported to be required for Cx43CT-ZO-1 binding. The c-Src SH3 domain affected Cx43CT residues Lys-264 -Lys-287, Ser-306 -Glu-316, His-331-Phe-337, Leu-356 -Val-359, and Ala-367-Ser-372. Only region Lys-264 -Lys-287 contains the residues previously reported to act as an SH3 binding domain. The specificity of these interactions was verified by peptide competition experiments. Finally, we demonstrated that the SH3 domain could partially displace the Cx43CT-PDZ-2 complex. These studies represent the first structural characterization of a connexin domain when integrated in a multimolecular complex. Furthermore, we demonstrate that the structural characteristics of a disordered Cx43CT are advantageous for signaling between different binding partners that may be important in describing the mechanism of channel closure or internalization in response to pathophysiological stimuli.Gap junction channels serve to directly interconnect the cytoplasm of neighboring cells, allowing the passage of moderately small ions, metabolites, and signaling molecules. Mammalian gap junction channels are formed by as many as 21 different connexin proteins (1). Of these, connexin43 (Cx43) 1 is the most completely characterized in terms of channel gating properties (2-4), phosphorylation sites (5-7), mechanisms of pH sensitivity (8 -11), and overall molecular structure (12). Cx43 is the most abundant gap junction protein in various tissues, including heart and brain. Cx43 null mice have been extensively investigated, with important differences being found as compared with wild types with regard to numerous processes, including cardiac developmental abnormalities, electrical synchrony in the heart, spreading depression in brain, as well as global gene expression changes in heart and astrocytes (13)(14)(15)(16)(17)(18)(19)(20).Connexin molecules are tetraspan membrane proteins, with both amino and carboxyl termini within the cytoplasm. Although the structure of the membrane-spanning portions of Cx43 has been solved to a resolution of about 7.5 Å (in the membrane plane) using electron crystallography (12), a constr...

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...

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