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Astrocytic gap junctions close in response to low intracellular pH, a process that may occur during ischemia. We propose that pH gating of Connexin43 (Cx43) involves binding of the carboxyl terminal domain (Cx43CT) to a pore-affiliated ‘receptor’ and, possibly, interaction of Cx43 with other molecular partners. We studied the role of intra– and intermolecular interactions in Cx43 pH gating. Peptides were made corresponding to intracellular regions of Cx43. Using Surface Plasmon resonance (SPR) we found pH dependent binding of the second half of the cytoplasmic loop to Cx43CT (Kd = 80 and 225 mm, pH 6.5 and 7.4, respectively). Binding was inhibited by preincubation with a synthetic peptide from region 346–362 of Cx43CT and by an antibody to this epitope, suggesting the binding site lies partially within this region. Nuclear magnetic resonance (NMR) studies showed formation of alpha helices in response to low pH in amino acid sequences VEMHL (aa 123–127) and IEEHGK (aa 139–143) in the loop, and a helical region within the suggested Cx43CT binding site. In addition, we found that acidification of astrocytes in vitro led to dissociation of Cx43 from its scaffold protein ZO-1. SPR showed that this was not due to pH dependence of Cx43-ZO-1 interaction (Kd = 2.23 mm at pH 7.4, 1.71 mm at pH 6.5) but to a strong pH–dependent interaction of Cx43 with c-Src (Kd = 0.63 mm at pH 7.4, 0.04 mm at pH 6.5) which caused ZO-1 to dissociate from Cx43CT. Separate experiments showed activation of astrocytic src at low pH. These changes in the composition of the Cx43 Nexus may play a major role in limiting tissue damage during brain ischemia. Acknowledgements: Supported by NIH grants NS 07098 (HSD) NS34931 & NS41282 (DCS) and GM57691 (MD).

Connexins proteins associate with a variety of catalytic and non-catalytic molecules. Also, different domains of connexin can bind to each other, providing a mechanism for channel regulation. Here, we review some of these associations, placing particular emphasis on the intramolecular interactions that regulate Connexin43 (Cx43). We also describe some novel methods that allow for the characterization of protein-protein interactions such as those observed in the cardiac gap junction protein Connexin43. Overall, intra- and inter-molecular interactions may regulate gap junctions to filter the passage of molecular messages between cells at the appropriate time and between the appropriate cells. As a potential area for future investigations, we also speculate as to whether some of the inter-molecular interactions involving connexins lead to modifications in the function of the associated protein, rather than on the function of connexin itself.

Determination of the protein-protein interactions of connexins has become a rapidly expanding field of research. While there are multiple methods of determining the identity of binding partners, determination of the strengths of interactions is not as simple. Here we describe the use of the in vitro method Enzyme Linked Sorbent Assay (ELSA) to compare binding affinities of known protein partners for Connexin43. We used the binding of Cx43 Carboxyl Terminal domain to the PDZ-2 domain of Zonula Occludens-1 and to the SH3 domain of c-Src. In the ELSA assay we found that while the binding of the SH3 domain of c-Src is pH-dependent, the interaction of the PDZ domain of ZO-1 is not. These data confirm findings using Surface Plasmon Resonance (1) and indicate that ELSA can be a useful tool in determining the kinetics of protein-protein interactions.

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 alpha-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.

Gap junction channels allow for the passage of ions and small molecules between neighboring cells. These channels are formed by multimers of an integral membrane protein named connexin. In the heart and other tissues, the most abundant connexin is a 43-kDa, 382-amino acid protein termed connexin43 (Cx43). A characteristic property of connexin channels is that they close upon acidification of the intracellular space. Previous studies have shown that truncation of the carboxyl terminal of Cx43 impairs pH sensitivity. In the present study, we have used a combination of optical, electrophysiological, and molecular biological techniques and the oocyte expression system to further localize the regions of the carboxyl terminal that are involved in pH regulation of Cx43 channels. Our results show that regions 261-300 and 374-382 are essential components of a pH-dependent "gating particle," which is responsible for acidification-induced uncoupling of Cx43-expressing cells. Regions 261-300 and 374-382 seem to be interdependent. The function of region 261-300 may be related to the presence of a poly-proline repeat between amino acids 274 and 285. Furthermore, site-directed mutagenesis studies show that the function of region 374-382 is not directly related to its net balance of charges, although mutation of only one amino acid (aspartate 379) for asparagine impairs pH sensitivity to the same extent as truncation of the carboxyl terminal domain (from amino acid 257). The mutation in which serine 364 is substituted for proline, which has been associated with some cases of cardiac congenital malformations in humans, also disrupts the pH gating of Cx43, although deletion of amino acids 364-373 has no effect on acidification-induced uncoupling. These results provide new insight into the molecular mechanisms responsible for acidification-induced uncoupling of gap junction channels in the heart and in other Cx43-expressing structures.

Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called “RXP-E” binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP–RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing ≈20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP–RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pH i 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP–RXP-E–treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart.

Migration of the gap junction protein connexin 43 (Cx43) in SDS-PAGE yields 2 to 4 distinct bands, detectable in the 40-47 kDa range. Here, we show that antibodies against the carboxy-terminal domain of Cx43 recognized an additional 20-kDa product. This protein was detected in some culture cell lysates. The presence of the 20-kDa band was not prevented by the use of protease inhibitors (Complete(R) and phenylmethylsulfonyl fluoride (PMSF), 1-5 mM). The band was absent from cells treated with Cx43-specific RNAi, and from those derived from Cx43-deficient mice, indicating that this Cx43-immunoreactive protein is a product of the Cx43 gene. Treatment of CHO cells with cyclosporin A caused a reduction in the amount of full-length Cx43 and a concomitant increase in the amount of the 20-kDa band. Overall, our data show that a fraction of the Cx43-immunoreactive protein pool within a given cell may correspond to a C-terminal fragment of the protein.

The carboxyl-terminal domain of connexin43 (Cx43CT) is involved in various intra- and intermolecular interactions that regulate gap junctions. Here, we used phage display to identify novel peptidic sequences that bind Cx43CT and modify Cx43 regulation. We found that Cx43CT binds preferentially to peptides containing a sequence RXP, where X represents any amino acid and R and P correspond to the amino acids arginine and proline, respectively. A biased “RXP library” led to the identification of a peptide (dubbed “RXP-E”) that bound Cx43CT with high affinity. Nuclear magnetic resonance data showed RXP-E–induced shifts in the resonance peaks of residues 343 to 346 and 376 to 379 of Cx43CT. Patch-clamp studies revealed that RXP-E partially prevented octanol-induced and acidification-induced uncoupling in Cx43-expressing cells. Moreover, RXP-E increased mean open time of Cx43 channels. The full effect of RXP-E was dependent on the integrity of the CT domain. These data suggest that RXP-based peptides could serve as tools to help determine the role of Cx43 as a regulator of function in conditions such as ischemia-induced arrhythmias.

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 (approximately 100 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 alpha-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.

Objectives The aim of this study was to determine if the structural integrity of a region in the cytoplasmic loop (amino acids 119–144; region “L2”) of connexin43 (Cx43) is necessary to maintain normal channel function. Background Cx43 is the most abundant gap junction protein in the heart. The ability of these channels to close under pathologic conditions such as ischemia may be a key substrate for cardiac arrhythmias. Previous studies have shown that Cx43 regulation involves the intramolecular interaction of its carboxyl terminal domain (a “gating particle”) with a separate region of the molecule acting as a receptor. We recently proposed that a region in the cytoplasmic loop of Cx43 (amino acids 119–144; region “L2”) might function as a receptor. Methods Using site-directed mutagenesis and patch clamp analysis, as well as fluorescent microscopy, we examined gap junction plaque formation and channel properties of Cx43 L2 mutants. Results Deletions of 5 to 6 amino acids within the L2 domain interfered with the formation of functional gap junction channels, although gap junction plaques were clearly visible. Selected point mutations in the region (including those present in patients with oculodentodigital dysplasia) caused modifications ranging from complete channel closure to changes in unitary conductance. Conclusions These results show that the L2 region is essential for maintenance of the normal architecture of the channel pore. This information is consistent with the notion that the L2 region could be a receptor for the carboxy terminal domain; the latter interaction would lead to channel closure under conditions such as myocardial ischemia and infarction.

Ischemia-induced acidification of astrocytes or cardiac myocytes reduces intercellular communication by closing gap junction channels and subsequently internalizing gap junction proteins. To determine whether such coupling changes might be attributable to altered interactions between connexin43 (Cx43) and other proteins, we applied the nigericin/high K + method to vary intracellular pH (pHi) in cultured cortical astrocytes. Intracellular acidification was accompanied by internalization of Cx43 with retention of Cx43 scaffolding protein Zonula Occludens-1 (ZO-1) at cell surfaces, suggesting that ZO-1 and Cx43 dissociate at low pHi. Coimmunoprecipitation studies revealed decreased binding of ZO-1 and increased binding of c-Src to Cx43 at low pHi. Resonant mirror spectroscopy was used to quantify binding of the SH3 domain of c-Src and the PDZ domains of ZO-1 to the carboxyl terminal domain of Cx43 (Cx43CT). Data indicate that the c-Src/Cx43CT interaction is highly pH dependent whereas the ZO-1/Cx43CT interaction is not. Moreover, binding of c-Src to Cx43CT prevented and reversed ZO-1/Cx43CT binding. We hypothesize that increased affinity of c-Src for Cx43 at low pHi aids in separation of Cx43 from ZO-1 and that this may facilitate internalization of Cx43. These data suggest that protracted acidification may remodel protein-protein interactions involving Cx43 and thus provide an important protective mechanism to limit lesion spread after ischemic injury.

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.

The gap junction family of integral membrane proteins enables the direct cytoplasmic exchange of ions and small molecules (

Connexins are integral membrane proteins that oligomerize to form intercellular gap junction channels. These channels allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. Many factors have been identified to regulate the channel gating of the major cardiac connexin, connexin43 (Cx43) and our laboratory has focused on pH-regulation, which is a cause of ischemia-induced arrhythmias. Our previous studies have suggested that regulation of Cx43 channels results from the association of the carboxyl-terminal domain (Cx43CT), acting as a gating particle, and the cytoplasmic loop domain (Cx43CL), acting as a receptor for the gating particle. Recently, we have identified a synthetic peptide, RXP-E (30-mer peptide containing a RXP sequence specifically recognized by Cx43CT, where X represents any amino acid, and R and P correspond to arginine and proline) that interacts with the Cx43CT and can prevent closure channel. The question remains as to whether this peptide is involved in the disruption of the Cx43CT/Cx43CL interaction. Using NMR, we investigated the changes in the structure of Cx43CT with RXP-E and identified the binding site in the first half of the Cx43CT. The identification of this new specific binding domain was furthermore confirmed by mutagenesis. Additionally, we studied the interaction of Cx43CT with Cx43CL and identified a similar binding region as RXPE, confirming the potentiality of our peptide to act as a “scaffold”, thereby holding the channels in its open state and reducing life threatening arrhythmias.