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...

pH-induced closure of connexin43 (Cx43) channels involves interaction of the Cx43 carboxyl-terminal (Cx43CT) with a separate "receptor" domain. The receptor location and structure and whether the interaction is directly intramolecular are unknown. Here we show resonant mirror technology, enzyme-linked sorbent assays, and nuclear magnetic resonance (NMR) experiments demonstrating pH-dependent binding of Cx43CT to region 119 -144 of Cx43 (Cx43L2), which we propose is the receptor. NMR showed that acidification induced ␣-helical order in Cx43L2, whereas only a minor modification in Cx43CT structure was detected. These data provide the first demonstration of chemically induced structural order and binding between cytoplasmic connexin domains.

Phosphorylation at unspecified sites is known to regulate the life cycle (assembly, gating, and turnover) of the gap junction protein, Cx43. In this paper, we show that Cx43 is phosphorylated on S365 in cultured cells and heart tissue. Nuclear magnetic resonance structural studies of the C-terminal region of Cx43 with an S365D mutation indicate that it forms a different stable conformation than unphosphorylated wild-type Cx43. Immunolabeling with an antibody specific for Cx43 phosphorylated at S365 shows staining on gap junction structures in heart tissue that is lost upon hypoxia when Cx43 is no longer specifically localized to the intercalated disk. Efficient phosphorylation at S368, an important Cx43 channel regulatory event that increases during ischemia or PKC activation, depends on S365 being unphosphorylated. Thus, phosphorylation at S365 can serve a “gatekeeper” function that may represent a mechanism to protect cells from ischemia and phorbol ester-induced down-regulation of channel conductance.

Structural information on membrane proteins lags far behind that on soluble proteins, in large part due to difficulties producing homogeneous, stable, structurally relevant samples in a membrane-like environment. In this study 25 membrane mimetics were screened using 2D (1)H-(15)N heteronuclear single quantum correlation NMR experiments to establish sample homogeneity and predict fitness for structure determination. A single detergent, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG), yielded high quality NMR spectra with sample lifetimes greater than one month for the five proteins tested - R. sphaeroides LH1 alpha and beta subunits, E. coli and B. pseudofirmus OF4 ATP synthase c subunits, and S. aureus small multidrug resistance transporter - with 1, 2, or 4 membrane spanning alpha-helices, respectively. Site-specific spin labeling established interhelical distances in the drug transporter and genetically fused dimers of c subunits in LPPG consistent with in vivo distances. Optical spectroscopy showed that LH1 beta subunits form native-like complexes with bacteriochlorophyll a in LPPG. All the protein/micelle complexes were estimated to exceed 100 kDaltons by translational diffusion measurements. However, analysis of (15)N transverse, longitudinal and (15)N[(1)H] nuclear Overhauser effect relaxation measurements yielded overall rotational correlation times of 8 to 12 nsec, similar to a 15-20 kDalton protein tumbling isotropically in solution, and consistent with the high quality NMR data observed.

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.