Search

Rationale: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (Na V 1.5) to this region. Objective: To establish the association of PKP2 with sodium channels and its role on action potential propagation. Methods and Results: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with Na V 1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. Conclusions: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed.

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).

Background Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by ventricular arrhythmias, sudden death, and fatty or fibrofatty replacement of right ventricular myocytes. Recent studies have noted an association between human ARVD/C and molecular remodeling of intercalated disc structures. However, progress has been constrained by limitations inherent to human studies. Objective We studied the molecular composition of the intercalated disc structure in a naturally occurring animal model of ARVD/C (Boxer dogs). Methods We studied hearts from 12 Boxers with confirmed ARVD/C and 2 controls. Ventricular sections from 4 animals were examined by immunofluorescent microscopy. Frozen tissue samples were used for Western blot analysis. Proteins investigated were N-cadherin, plakophilin 2, desmoplakin, plakoglobin, desmin, and connexin 43 (Cx43). Results In control dogs, all proteins tested by immunofluorescence analysis yielded intense localized signals at sites of end-to-end cell apposition. In contrast, myocardial tissues from ARVD/C-afflicted Boxers showed preservation of N-cadherin staining but loss of detectable signal for Cx43 at the intercalated disc location. Western blots indicated that the Cx43 protein was still present in the samples. Gene sequencing analysis showed no mutations in desmoplakin, plakoglobin, Cx43, or plakophilin 2. Conclusion Mutation(s) responsible for ARVD/C in Boxers lead, directly or indirectly, to severe modifications of mechanical and electrical cell–cell interactions. Furthermore, significant reduction in gap junction formation may promote a substrate for malignant ventricular arrhythmias. This model may help to advance our understanding of the molecular basis, pathophysiology, and potential therapeutic approach to patients with ARVD/C.

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.

Background Gap junctions are potential targets for pharmacologic intervention. We previously developed a series of peptide sequences that prevent closure of connexin43 (Cx43) channels, bind to cardiac Cx43, and prevent acidification-induced uncoupling of cardiac gap junctions. Objective The purpose of this study was to identify and validate the minimum core active structure in peptides containing an RR-N/Q-Y motif. Based on that information, we sought to generate a peptidomimetic molecule that acts on the chemical regulation of Cx43 channels. Methods Experiments were based on a combination of biochemical, spectroscopic, and electrophysiologic techniques as well as molecular modeling of active pharmacophores with Cx43 activity. Results Molecular modeling analysis indicated that the functional elements of the side chains in the motif RRXY form a triangular structure. Experimental data revealed that compounds containing such a structure bind to Cx43 and prevent Cx43 chemical gating. These results provided us with the first platform for drug design targeted to the carboxyl terminal of Cx43. Using that platform, we designed and validated a peptidomimetic compound (ZP2519; molecular weight 619 Da) that prevented octanol-induced uncoupling of Cx43 channels and pH gating of cardiac gap junctions. Conclusion Structure-based drug design can be applied to the development of pharmacophores that act directly on Cx43. Small molecules containing these pharmacophores can serve as tools to determine the role of gap junction regulation in the control of cardiac rhythm. Future studies will determine whether these compounds can function as pharmacologic agents for the treatment of a selected subset of cardiac arrhythmias.

Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-aa peptide (RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development, remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear magnetic resonance and patch-clamp strategies to define, for the first time, a unique ensemble of pharmacophores that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this family: a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY. These 2 small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers. Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function.

The physiology of gap junctions is regulated by changes in the microenvironment of the cell. The most defined mechanism for such regulation is the closure of gap junction channels in response to acidification of the intracellular space, that is, pH gating or chemical gating. In the best studied case, that of Cx43, chemical gating involves interactions between a specific segment of the cytoplasmic carboxyl-terminal domain with a specific segment of the cytoplasmic loop domain. These interactions have been characterized at the molecular and structural levels. Additionally, there is evidence that interaction of cytoplasmic aminosulfonates and peptide reagents can also affect the gating mechanisms controlled by these inter-domain interactions.

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.

Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to mutations in desmosomal proteins, including plakophilin-2 (PKP2). Little is known about the changes in cellular function and structure that follow expression of ARVC-relevant PKP2 mutations. Objective The purpose of this study was to investigate the function and distribution of an ARVC-relevant PKP2 mutant where arginine at position 79 was replaced by a stop codon (R79x). Methods Results were compared with those obtained with mutation 179fs (frameshift at position 179). Mutant constructs were introduced by adenoviral infection into neonatal rat ventricular myocytes in culture. Results Both mutant proteins failed to preferentially localize to sites of cell-cell apposition. Their expression did not disrupt localization of endogenous PKP2, connexin-43 (Cx43), or desmoplakin (DP). However, we observed reduced abundance of Cx43 after R79x expression. Early truncation of PKP2 at position 79 also prevented its physical interaction with both DP and Cx43. Finally, R79x expression correlated with loss of expression of HSP90, a protein relevant to cardiomyocyte apoptosis. Conclusion These results provide the first observations of the cellular/molecular phenotype consequent to these PKP2 mutations and give insight into the possible cellular substrates that lead to ARVC.

Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts. We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins. The results are discussed in the context of arrhythmogenic right ventricular cardiomyopathy, an inherited disease involving mutations in desmosomal proteins, including PKP2.

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.

The topic of protein-protein interactions has become a major research area in recent years. The reductionist approach of identifying individual genes and proteins is now being matched by the analysis of the interactions that individual players undergo. One could intuitively argue that molecules in a cell are absent of function if they cannot associate and interact with others. The product of a gene is a character in a play, not the play itself. It is in the way the players interact that a function is achieved. Here, we review the basic theory and some of the practical aspects in the application of a technique called “surface plasmon resonance” (SPR) to study intermolecular interactions in vitro.

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.

Connexin43 (Cx43) channels reside in at least 3 states: closed, open, or residual. It is hypothesized that the residual state results from the interaction of an intracellular “gating element” with structures at the vestibule of the pore. Recently, we showed in vitro that there is an intramolecular interaction of the carboxyl-terminal domain (referred to as “CT”) with a region in the cytoplasmic loop of Cx43 (amino acids 119 to 144; referred to as “L2”). Here, we assessed whether the L2 region was able to interact with the gating particle in a functional channel. Cx43 channels were recorded in the presence of a peptide corresponding to the L2 region, delivered via the patch pipette. This manipulation did not modify unitary conductance, but decreased the frequency of transitions into the residual state, prolonged open time, and altered the voltage dependence of the channel in a manner analogous to that observed after truncation of the CT domain. The latter correlated with the ability of the peptide to bind to the CT domain, as determined by mirror resonance spectroscopy. Overall, we propose that the L2 acts as a “receptor” that interacts with a flexible intracellular gating element during channel gating. The full text of this article is available online at http://circres.ahajournals.org.

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.

Abstract —Connexin43(Cx43) channels can be regulated by a variety of factors, including low pH i . Structure/function studies from this laboratory have demonstrated that pH gating follows a particle-receptor mechanism, similar to the “ball-and-chain” model of voltage-dependent inactivation of ion channels. The question whether the particle-receptor model is applicable only to pH gating or to other forms of Cx43 regulation as well remains. To address this question, we looked at the uncoupling effects of insulin and of insulin-like growth factor-1 (IGF) on Cx43 channels expressed in Xenopus oocytes. These agonists do not induce changes in pH i . Junctional conductance (Gj) was measured by the dual 2-electrode voltage-clamp technique. Control studies showed that relative Gj did not change spontaneously as a function of time. Continuous exposure of Cx43-expressing oocytes to insulin (10 μ/L) led to a decrease in Gj. After 80 minutes, Gj was 54±5% from control (n=12). Exposure of oocytes to IGF (10 nmol/L) caused an even more pronounced change in Gj (37±4% of control, n=6). The time course of the IGF-induced uncoupling was similar to that observed after insulin exposure. The effect of insulin was abolished by truncation of the carboxyl-terminal domain of Cx43 at amino acid 257 (M257). Interestingly, as in the case of pH gating, coexpression of the carboxyl-terminal domain (amino acids 258 to 282) together with M257 rescued the ability of insulin to reduce coupling (Gj, 39±12% from control; n=6). Structure/function experiments using various deletion mutants of the carboxyl-terminal domain showed that insulin treatment does not modify Gj if amino acids 261 to 280 are missing from the Cx43 sequence. Our results suggest that a particle-receptor (or ball-and-chain) mechanism, similar to that described for pH gating, also applies to chemical regulation of Cx43 by other factors.

Myocarditis, or inflammation of the heart muscle, is associated with severe morbidity and mortality, even in young, otherwise healthy individuals. The disease is initiated by exposure to viruses, parasites, and toxins, but can develop into a chronic autoimmune disease which progresses to severe fibrosis and dilated cardiomyopathy. Work in our lab using experimental autoimmune myocarditis (EAM) as a model, has demonstrated alterations in the sub-cellular localization of the cardiac gap junction protein connexin43 (Cx43) in heart tissue adjacent to myocarditis lesions, a potentially arrythmogenic effect. In the present study, we found the converse to be true; a reduction in Cx43 expression altered the course of EAM, resulting in a more pronounced disease. To generate animals with reduced Cx43, we backcrossed CX43+/- mice on the EAM-resistant C57BL/6 background to the EAM-susceptible BALB/c background. After 8 generations, we initiated EAM by two doses of α myosin heavy chain antigenic peptide at days 0 and 7. Hearts were analyzed 21 days after EAM induction for both the area and intensity of immune infiltrate. We determined that mice heterozygous for Cx43 develop more severe disease than wild type littermates. Closer analysis of infiltrating immune cells by immunofluorescence microscopy revealed differences in myeloperoxidase positive and CD3+ cells, with Cx43 heterozygotes having more of both. These results suggest that Cx43 can alter the course of EAM.

Gap Junction Proteins in the Sinoatrial Node. Introduction: Gap junction proteins are thought to form the low resistance pathways that connect neighboring cells within the sinoatrial node, and to mediate pacemaker synchronization. Methods and Results: We have carried out microelectrode mapping experiments of the hamster sinoatrial region to localize the primary pacemaker area for subsequent light, electron, and immunofluorescence microscopic studies aimed at testing the hypothesis that the major cardiac gap junction protein (connexin43) is present in such an area. The site of earliest activation is unifocal and the pattern of activation, obtained In multiple sequential microelectrode recordings of the Sinoatrial region, is qualitatively similar to that previously described for other species. However, quantitatively, the impulse transmission time from the primary pacemaker area to the crista (sulcus) terminal's in the hamster sinoatrial node is about 50% briefer than that of the guinea pig and five times faster than that of the rabbit. Immunolocalization studies in the hamster sinoatrial node using anti-connexin43 antisera demonstrated specific staining at the areas of cell-to-cell apposition and suggested that the apparently high degree of electrical coupling in this tissue is the result of abundant connexin43 expression. The immunofluorescence data were supported by light microscopic studies, which demonstrated the typical morphologic characteristics of sinus nodal cells in the pacemaker area. In addition, an electron microscopic study of the sinoatrial region revealed the presence of gap junctions in the junctional complex at areas of cell-to-cell contact. Conclusion: Our results demonstrate that cells in the sinoatrial region of the hamster heart are electrically well coupled and strongly suggest that such coupling is mediated by gap junctional channels formed by connexin43.

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.

www.elsevier.com/locate/jelectrocard Manipulation of gap junction proteins: effect on cellular coupling and impulse propagation S.M. Taffet, S. Shah, R.L. Lewandowski, W. Coombs J. Shibayama, P. Sorgen, M. Delmar (Upstate Medical University, Syracuse, NY, USA; University of Nebraska, Omaha, NE, USA) The cardiac gap junction protein, connexin43 (Cx43), is not a passive pore but an actively regulated channel. Under conditions of cellular stress, such as acidification, the pore closes resulting in alterations in impulse propagation. We have previously demonstrated that the carboxyl terminal (CT) domain of Cx43 (Cx43CT) is involved in various intraand intermolecular interactions that regulate gap junctions. Here, we used a nonbiased phage display library to identify novel peptidic sequences that bind Cx43CT and hopefully will alter the properties of the channel. A library of phage containing a randomized series of 12–amino acid peptides (approximately 10 different sequences) was presented to immobilized Cx43CT (pH, 6.5). Analysis of the selected phage revealed a total of 48 Cx43CTinteracting peptides. One third of the peptides had a preserved arginine-X-proline (RXP) sequence. Surface plasmon resonance confirmed binding of 6 of these peptides to Cx43CT. Two of these peptides (RXP-1 and RXP-4) were further analyzed. The interaction of RXP-4 with the CX43-CT was further analyzed by NMR revealing 0022-0736/$ – see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.jelectrocard.2005.06.091 that this peptide modifies the structure of region 375-379 of Cx43CT. Dual-patch clamp analysis revealed that RXP-4 (but not RXP-1) reduced the frequency with which Cx43 channels transit into the residual state. Unitary conductances were not modified by this peptide. To further analyze the optimal RXP sequences for binding and modifying the function of Cx43, we generated a biased phage display library where the RXP sequence was flanked on each side by 4 random amino acids. Approximately 40000 distinct sequences were presented to Cx43CT. A minor fraction of the inserts resulted from concatenation of 2 peptides in tandem (a bdoublet Q). No doublets were found in a random sampling of 63 inserts obtained before binding. Yet, of the 485 clones sequenced after selection by binding, 85% were doublets, suggesting a high selectivity of Cx43CT for doublets during the binding step. This increased affinity of doublets for CT interaction was confirmed by surface plasmon resonance. Further analysis of the sequences obtained by phage display indicated that there was an increased occurrence of acidic amino acids at position 3 (relative to RXP) but an absence of acidic residues at positions 2 and +1. Basic amino acids were absent from position 1. Overall, these data show that RXP peptides can lead to structural modifications in Cx43CT and changes in the function of Cx43 channels. These results open the door for further development of peptide-based strategies to modify the regulation of Cx43-containing gap junctions in native tissues such as heart. ardiology 38 (2005) 51