A Modified Tridecapeptide Probe for Imaging Cell Junction.

Cell junctions, which are typically associated with dynamic cytoskeletons, are essential for a wide range of cellular activities, including cell migration, cell communication, barrier function and signal transduction. Observing cell junctions in real-time can help us understand the mechanisms by which they regulate these cellular activities. This study examined the binding capacity of a modified tridecapeptide from Connexin 43 (Cx43) to the cell junction protein zonula occludens-1 (ZO-1). The goal was to create a fluorescent peptide that can label cell junctions. A cell-penetrating peptide was linked to the modified tridecapeptide. The heterotrimeric peptide molecule was then synthesized. The binding of the modified tridecapeptide was tested using pulldown and immunoprecipitation assays. The ability of the peptide to label cell junctions was assessed by adding it to fixed or live Caco-2 cells. The testing assays revealed that the Cx43-derived peptide can bind to ZO-1. Additionally, the peptide was able to label cell junctions of fixed cells, although no obvious cell junction labeling was observed clearly in live cells, probably due to the inadequate affinity. These findings suggest that labeling cell junctions using a peptide-based strategy is feasible. Further efforts to improve its affinity are warranted in the future.

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure.

BACKGROUND: Atrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. OBJECTIVES: In this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. METHODS: The authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF. RESULTS: AF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with NaV1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1. CONCLUSIONS: These results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.

Interrogation of Carboxy-Terminus Localized GJA1 Variants Associated with Erythrokeratodermia Variabilis et Progressiva.

Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell-cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.

Harnessing the therapeutic potential of antibodies targeting connexin hemichannels.

BACKGROUND: Connexin hemichannels have been implicated in pathology-promoting conditions, including inflammation, numerous widespread human diseases, including cancer and diabetes, and several rare diseases linked to pathological point mutations. METHODS: We analysed the literature focusing on antibodies capable of modulating hemichannel function, highlighting generation methods, applications to basic biomedical research and translational potential. RESULTS: Anti-hemichannel antibodies generated over the past 3 decades targeted mostly connexin 43, with a focus on cancer treatment. A slow transition from relatively unselective polyclonal antibodies to more selective monoclonal antibodies resulted in few products with interesting characteristics that are under evaluation for clinical trials. Selection of antibodies from combinatorial phage-display libraries, has permitted to engineer a monoclonal antibody that binds to and blocks pathological hemichannels formed by connexin 26, 30 and 32. CONCLUSIONS: All known antibodies that modulate connexin hemichannels target the two small extracellular loops of the connexin proteins. The extracellular region of different connexins is highly conserved, and few residues of each connexins are exposed. The search for new antibodies may develop an unprecedented potential for therapeutic applications, as it may benefit tremendously from novel whole-cell screening platforms that permit in situ selection of antibodies against membrane proteins in native state. The demonstrated efficacy of mAbs in reaching and modulating hemichannels in vivo, together with their relative specificity for connexins overlapping epitopes, should hopefully stimulate an interest for widening the scope of anti-hemichannel antibodies. There is no shortage of currently incurable diseases for which therapeutic intervention may benefit from anti-hemichannel antibodies capable of modulating hemichannel function selectively and specifically.

Gap19, a Cx43 Hemichannel Inhibitor, Acts as a Gating Modifier That Decreases Main State Opening While Increasing Substate Gating.

Cx43 hemichannels (HCs) are electrically and chemically gated transmembrane pores with low open probability and multiple conductance states, which makes kinetic studies of channel gating in large datasets challenging. Here, we developed open access software, named HemiGUI, to analyze HC gating transitions and investigated voltage-induced HC opening based on up to ≈4000 events recorded in HeLa-Cx43-overexpressing cells. We performed a detailed characterization of Cx43 HC gating profiles and specifically focused on the role of the C-terminal tail (CT) domain by recording the impact of adding an EGFP tag to the Cx43 CT end (Cx43-EGFP) or by supplying the Cx43 HC-inhibiting peptide Gap19 that interferes with CT interaction with the cytoplasmic loop (CL). We found that Gap19 not only decreased HC opening activity to the open state (≈217 pS) but also increased the propensity of subconductance (≈80 pS) transitions that additionally became slower as compared to the control. The work demonstrates that large sample transition analysis allows detailed investigations on Cx43 HC gating and shows that Gap19 acts as a HC gating modifier by interacting with the CT that forms a crucial gating element.

Inhibition of Pyk2 and Src activity improves Cx43 gap junction intercellular communication.

Identification of proteins that interact with Cx43 has been instrumental in the understanding of gap junction (GJ) regulation. An in vitro phosphorylation screen identified that Protein tyrosine kinase 2 beta (Pyk2) phosphorylated purified Cx43CT and this led us to characterize the impact of this phosphorylation on Cx43 function. Mass spectrometry identified Pyk2 phosphorylates Cx43 residues Y247, Y265, Y267, and Y313. Western blot and immunofluorescence staining using HeLaCx43 cells, HEK 293 T cells, and neonatal rat ventricular myocytes (NRVMs) revealed Pyk2 can be activated by Src and active Pyk2 interacts with Cx43 at the plasma membrane. Overexpression of Pyk2 increases Cx43 phosphorylation and knock-down of Pyk2 decreases Cx43 phosphorylation, without affecting the level of active Src. In HeLaCx43 cells treated with PMA to activate Pyk2, a decrease in Cx43 GJ intercellular communication (GJIC) was observed when assayed by dye transfer. Moreover, PMA activation of Pyk2 could be inhibited by the small molecule PF4618433. This partially restored GJIC, and when paired with a Src inhibitor, returned GJIC to the no PMA control-level. The ability of Pyk2 and Src inhibitors to restore Cx43 function in the presence of PMA was also observed in NRVMs. Additionally, an animal model of myocardial infarction induced heart failure showed a higher level of active Pyk2 activity and increased interaction with Cx43 in ventricular myocytes. Src inhibitors have been used to reverse Cx43 remodeling and improve heart function after myocardial infarction; however, they alone could not fully restore proper Cx43 function. Our data suggest that Pyk2 may need to be inhibited, in addition to Src, to further (if not completely) reverse Cx43 remodeling and improve intercellular communication.

Canonical and Non-Canonical Roles of Connexin43 in Cardioprotection.

Since the mid-20th century, ischemic heart disease has been the world's leading cause of death. Developing effective clinical cardioprotection strategies would make a significant impact in improving both quality of life and longevity in the worldwide population. Both ex vivo and in vivo animal models of cardiac ischemia/reperfusion (I/R) injury are robustly used in research. Connexin43 (Cx43), the predominant gap junction channel-forming protein in cardiomyocytes, has emerged as a cardioprotective target. Cx43 posttranslational modifications as well as cellular distribution are altered during cardiac reperfusion injury, inducing phosphorylation states and localization detrimental to maintaining intercellular communication and cardiac conduction. Pre- (before ischemia) and post- (after ischemia but before reperfusion) conditioning can abrogate this injury process, preserving Cx43 and reducing cell death. Pre-/post-conditioning has been shown to largely rely on the presence of Cx43, including mitochondrial Cx43, which is implicated to play a major role in pre-conditioning. Posttranslational modifications of Cx43 after injury alter the protein interactome, inducing negative protein cascades and altering protein trafficking, which then causes further damage post-I/R injury. Recently, several peptides based on the Cx43 sequence have been found to successfully diminish cardiac injury in pre-clinical studies.

Antagonistic Functions of Connexin 43 during the Development of Primary or Secondary Bone Tumors.

Despite research and clinical advances during recent decades, bone cancers remain a leading cause of death worldwide. There is a low survival rate for patients with primary bone tumors such as osteosarcoma and Ewing's sarcoma or secondary bone tumors such as bone metastases from prostate carcinoma. Gap junctions are specialized plasma membrane structures consisting of transmembrane channels that directly link the cytoplasm of adjacent cells, thereby enabling the direct exchange of small signaling molecules between cells. Discoveries of human genetic disorders due to genetic mutations in gap junction proteins (connexins) and experimental data using connexin knockout mice have provided significant evidence that gap-junctional intercellular communication (Gj) is crucial for tissue function. Thus, the dysfunction of Gj may be responsible for the development of some diseases. Gj is thus a main mechanism for tumor cells to communicate with other tumor cells and their surrounding microenvironment to survive and proliferate. If it is well accepted that a low level of connexin expression favors cancer cell proliferation and therefore primary tumor development, more evidence is suggesting that a high level of connexin expression stimulates various cellular process such as intravasation, extravasation, or migration of metastatic cells. If so, connexin expression would facilitate secondary tumor dissemination. This paper discusses evidence that suggests that connexin 43 plays an antagonistic role in the development of primary bone tumors as a tumor suppressor and secondary bone tumors as a tumor promoter.

Amphiphilic aminoglycosides with increased selectivity for inhibition of connexin 43 (Cx43) hemichannels.

Gap junction channels formed by the association of connexin hemichannels play a crucial role in intercellular communication. Connexin 43 (Cx43) is expressed in a variety of tissues and organs, including heart and brain, and abnormal sustained opening of undocked "free" hemichannels contributes to the cell damage in cardiac infarcts and stroke. Selective inhibitors of Cx43 hemichannels for clinical use are then desirable. Here, we synthesized and tested new aminoglycosides for their connexin inhibitory activity towards Cx26 and Cx43 hemichannels. The lead compounds displayed enhanced Cx43/Cx26 selectivity for hemichannel inhibition when compared to the parent kanamycin A and other commercially available aminoglycosides. These lead compounds are not cytotoxic to mammalian cells and show promise for the treatment of ischemic damage of the heart, brain, and kidneys. We identified a new compound as a promising lead based on its good selectivity for Cx43 hemichannels inhibition and the simplicity and affordability of its production.

Cardiac remodeling and arrhythmogenesis are ameliorated by administration of Cx43 mimetic peptide Gap27 in heart failure rats.

Alterations in connexins and specifically in 43 isoform (Cx43) in the heart have been associated with a high incidence of arrhythmogenesis and sudden death in several cardiac diseases. We propose to determine salutary effect of Cx43 mimetic peptide Gap27 in the progression of heart failure. High-output heart failure was induced by volume overload using the arterio-venous fistula model (AV-Shunt) in adult male rats. Four weeks after AV-Shunt surgery, the Cx43 mimetic peptide Gap27 or scrambled peptide, were administered via osmotic minipumps (AV-ShuntGap27 or AV-ShuntScr) for 4 weeks. Cardiac volumes, arrhythmias, function and remodeling were determined at 8 weeks after AV-Shunt surgeries. At 8th week, AV-ShuntGap27 showed a marked decrease in the progression of cardiac deterioration and showed a significant improvement in cardiac functions measured by intraventricular pressure-volume loops. Furthermore, AV-ShuntGap27 showed less cardiac arrhythmogenesis and cardiac hypertrophy index compared to AV-ShuntScr. Gap27 treatment results in no change Cx43 expression in the heart of AV-Shunt rats. Our results strongly suggest that Cx43 play a pivotal role in the progression of cardiac dysfunction and arrhythmogenesis in high-output heart failure; furthermore, support the use of Cx43 mimetic peptide Gap27 as an effective therapeutic tool to reduce the progression of cardiac dysfunction in high-output heart failure.

Development of PLGA nanoparticles for sustained release of a connexin43 mimetic peptide to target glioblastoma cells.

Effective therapeutic delivery of peptide and protein drugs is challenged by short in vivo half-lives due to rapid degradation. Sustained release formulations of αCT1, a 25 amino acid peptide drug, would afford lower dosing frequency in indications that require long term treatment, such as chronic wounds and cancers. In this study, rhodamine B (RhB) was used as a model drug to develop and optimize a double emulsion-solvent evaporation method of poly(lactic-co-glycolic acid) (PLGA) nanoparticle synthesis. Encapsulation of αCT1 in these nanoparticles (NPs) resulted in a sustained in vitro release profile over three weeks, characterized by an initial burst release of approximately 50% of total encapsulated drug over the first three days followed by sustained release over the remaining two and a half weeks. NP uptake by glioblastoma stem cells was through endocytosis and RhB and αCT1 were observed in cells after at least 4 days.

Hypoxia­induced internalization of connexin 26 and connexin 43 in pulmonary epithelial cells is involved in the occurrence of non­small cell lung cancer via the P53/MDM2 signaling pathway.

Reports have highlighted an association between connexins (CXs) or gap junction proteins and non­small cell lung cancer (NSCLC). In the present study, it was aimed to elucidate the regulatory mechanism of CX26 and CX43 under hypoxic conditions in NSCLC. Clinical samples were collected for analysis of CX26 and CX43 expression and clinical cancerization followed by quantification of CX26 and CX43 expression. Following the establishment of an in vitro hypoxia model, P53/murine double minute­2 (MDM2) signaling pathway­, proliferation­ and epithelial­mesenchymal transition (EMT)­related genes were quantified to evaluate the influence of CX26 and CX43 on the biological functions of pulmonary epithelial cells in NSCLC. In addition, the proliferation and tumorigenicity of cancer cells were assessed by EdU staining and xenograft tumors, respectively. Decreased expression of CX26 and CX43 was found in cancer tissues compared with surrounding normal tissue. Hypoxia was shown to activate the P53/MDM2 axis and stimulate the downregulation, ubiquitination and degradation of CX26 and CX43, which were translocated from the membrane to the cytoplasm. Low levels of CX26 and CX43 were demonstrated to further promote EMT and the induction of the proliferation and tumorigenicity of cancer cells. These results were reflected by decreased E­cadherin expression and increased N­cadherin expression, along with increased cell migration, promoted cell proliferation ability and elevated relative protein expression of Oct4 and Nanog, and accelerated tumor growth, accompanied by a higher number of metastatic nodes. Taken together, the key observations of the present study demonstrate that the internalization of CX26 and CX43 promoted proliferation, EMT and migration and thus induced NSCLC via aberrant activation of the P53/MDM2 signaling pathway under hypoxic conditions.

Structural determinants underlying permeant discrimination of the Cx43 hemichannel.

Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichannel.

Cell-free synthesis of connexin 43-integrated exosome-mimetic nanoparticles for siRNA delivery.

Exosomes are naturally secreted nanovesicles that have emerged as a promising therapeutic nanodelivery platform, due to their specific composition and biological properties. However, challenges like considerable complexity, low isolation yield, drug payload, and potential safety concerns substantially reduce their pharmaceutical acceptability. Given that the nano-bio-interface is a crucial factor for nanocarrier behavior and function, modification of synthetic nanoparticles with the intrinsic hallmarks of exosomes' membrane to create exosome mimetics could allow for siRNA delivery in a safer and more efficient manner. Herein, connexin 43 (Cx43)-embedded, exosome-mimicking lipid bilayers coated chitosan nanoparticles (Cx43/L/CS NPs) were constructed by using cell-free (CF) synthesis systems with plasmids encoding Cx43 in the presence of lipid-coated CS NPs (L/CS NPs). The integration of de novo synthesized Cx43 into the lipid bilayers of L/CS NPs occurred cotranslationally during one-pot reaction and, more importantly, the integrated Cx43 was functionally active in transport. In addition to considerably lower cytotoxicity (

Connexin 43-serine 282 modulates serine 279 phosphorylation in cardiomyocytes.

Connexin 43 (Cx43) phosphorylation plays a pivotal role in cardiac electrical and contractile performance. In a previous study we have found that Cx43 phosphorylation at serine 282 (pS282) regulates cardiomyocyte survival. Considering that both sites are altered simultaneously in many studies, we designed this study to identify the status of S279 phosphorylation upon pS282 manipulation. In heterozygous mice with S282 gene substituted with alanine (S282A), we found ventricular arrhythmias with inhibition of Cx43 phosphorylation at both S282 and S279 in the hearts. In cultured neonatal rat ventricular myocytes (NRVMs), transfection of virus carrying S282A mutant also blocked Cx43 phosphorylation at both S279/282 and gap junction coupling, while expression of wild-type Cx43 or S279A did not. Further, NRVMs transfected with S282 phospho-mimicking mutant substituted with aspartate or treated with ATP exhibited promotions of Cx43 phosphorylation at S279/282 and intercellular communication. Therefore, this study demonstrated a regulatory role of Cx43-S282 on S279 phosphorylation in cardiomyocytes, and suggested an involvement of S279 in the Cx43-S282 mediated cardiomyocyte homeostasis.

Calcium-calmodulin gating of a pH-insensitive isoform of connexin43 gap junctions.

Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction communication and the synergism or antagonism between pH and Ca2+ has been questioned for decades. To assess the ability of Ca2+ ions to modulate Cx43 junctional conductance (gj) in the absence of pH-sensitivity, patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full-length Cx43 or the Cx43-M257 (Cx43K258stop) mutant protein, a carboxyl-terminus (CT) truncated version of Cx43 lacking pH-sensitivity. The addition of 1 µM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 gj to zero within 15 min of perfusion. This response was prevented by Ca2+-free saline or addition of 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. Internal addition of a connexin50 cytoplasmic loop calmodulin-binding domain (CaMBD) mimetic peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 µM Gap19 peptide had minimal effect. The investigation of the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 gj, but also noted the abolishment of the previously reported facilitated recovery of gj from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact in its absence. Sequence-specific connexin CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has negligible effect on this chemical gating mechanism.

Phosphorylation of Cx43 residue Y313 by Src contributes to blocking the interaction with Drebrin and disassembling gap junctions.

Phosphorylation regulates connexin43 (Cx43) function from assembly/disassembly to coupling at the plaque. Src is a tyrosine kinase known to both phosphorylate Cx43 (residues Y247 and Y265) and affect gap junction intercellular communication. However, the Cx43 carboxyl-terminal (CT) domain contains additional tyrosine residues and proteomic discovery mass spectrometry data identified Y313 as a potential phosphorylation target. Based upon the study of Lin et al. (2001) J. Cell Biol., which still observed tyrosine phosphorylation by Src when using a Cx43 Y247/Y265F mutant, we addressed the possibility of Y313 phosphorylation (pY313) by Src. In vitro Src phosphorylation of purified Cx43CT followed by mass spectroscopy revealed that Src also phosphorylates Y313. This observation was confirmed by repeating the in vitro phosphorylation using different combinations of Cx43CT Y → F mutants and a general anti-pTyr antibody. Next, a phospho-specific antibody was generated to help characterize the importance of pY313. We established an in cyto experimental system by stably expressing Cx43 WT and mutants (Y247F, Y265F, Y313F, Y247/265F, Y247/313F, Y265/313F, or Y247/265/313F) in Cx43-deficient HeLa cells. Cx43 WT and mutants, in the absence of v-Src, localized to the plasma membrane and formed gap junctions. When v-Src was over-expressed, Cx43 WT localized intracellularly, while all of the single and double mutants remained able to form plaques and transfer dye, albeit variable in number and amount, respectively. Complete Src-resistance was only achieved with the Cx43 Y247/265/313F mutant. Furthermore, Cx43 Y265F inhibited the ability of v-Src to phosphorylate Y247 and Y313 as well as phosphorylation at both Y265 and Y313 was necessary to inhibit the Cx43 interaction with Drebrin. Finally, we observed in diseased cardiac tissue, in which Src is active, an increase in intercalated disc and intracellular localized Cx43 pY313.

Connexin 43 (Cx43) in cancer: Implications for therapeutic approaches via gap junctions.

Gap junctions are membrane channels found in all cells of the human body that are essential to cellular physiology. Gap junctions are formed from connexin proteins and are responsible for transfer of biologically active molecules, metabolites, and salts between neighboring cells or cells and their extracellular environment. Over the last few years, aberrant connexin 43 (Cx43) expression has been associated with cancer recurrence, metastatic spread, and poor survival. Here we provide an overview of the general structure and function of gap junctions and review their roles in different cancer types. We discuss new therapeutic approaches targeting Cx43 and potential new ways of exploiting gap junction transfer for drug delivery and anti-cancer treatment. The permeability of Cx43 channels to small molecules and macromolecules makes them highly attractive targets for delivering drugs directly into the cytoplasm. Cancer cells overexpressing Cx43 may be more permeable and sensitive to chemotherapeutics. Because Cx43 can either act as a tumor suppressor or oncogene, biomarker analysis and a better understanding of how Cx43 contextually mediates cancer phenotypes will be required to develop clinically viable Cx43-based therapies.

Visualization of Annular Gap Junction Vesicle Processing: The Interplay Between Annular Gap Junctions and Mitochondria.

It is becoming clear that in addition to gap junctions playing a role in cell⁻cell communication, gap junction proteins (connexins) located in cytoplasmic compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unclear. In this study, immunocytochemical, super-resolution, and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structures and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures-annular gap junctions-were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest, among other possibilities, that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 delivery to the mitochondria. Furthermore, it points to the need for investigating annular gap junctions as more than only vesicles destined for degradation.