Gap Junction Plaques Research Articles

Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels

The three connexins expressed in the ocular lens each contain PDZ domain-binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell-cell communication in HeLa cells, whereas the addition of a seven-amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain-binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.

A novelGJA3mutation associated with congenital nuclear pulverulent and posterior polar cataract in a chinese family

Congenital cataract (CC) is the leading cause of visual disability in children. To date, mutations in many genes have been linked to CC. In a four-generation Chinese family with congenital nuclear pulverulent and posterior polar cataracts, we detected a heterozygous c.5G>A transition in the second exon of GJA3, resulting in the substitution of a highly conserved glycine with aspartic acid (p.G2D) at the N-terminus of the connexin46 (Cx46) protein. Wild type (wt) and mutant Cx46 plasmids were transfected into HeLa cells to examine the molecular basis of cataract formation. Unlike wt Cx46, Cx46G2D mutant formed gap junction plaques inefficiently, changed hemichannel permeability, and caused apoptosis. These results suggest that the glycine residue at the second position of the N-terminus is important for gap junction plaque formation and hemichannel function.

The gap junction life cycle

The gap junction life cycle

Gap junctions in human glioblastomas: implications for suicide gene therapy

Glioblastoma is a very aggressive astrocytic tumor and most patients have 1-year survival time after diagnosis. A promising therapeutic strategy is the local delivery of the herpes simplex virus thymidine kinase gene in the tumor bed followed by ganciclovir treatment. The presence of functional gap junctions is highly relevant for the success of suicide gene therapy. Connexins are expressed in practically all tissues and form gap junctions that allow intercellular communication. Connexin 43 (Cx43) is the major connexin member being expressed in astrocytes but its status in glioblastoma is not well defined. We have investigated by immunofluorescence the presence of Cx43 in 74 human glioblastoma samples; its expression was detected in 77% of the samples analyzed. We report here that glioblastoma is a heterogenous disease as regards Cx43 expression with presentations, in which Cx43 expression is unaltered, reduced or totally lost. A predominant Cx43 cytoplasmic localization was observed in four out of eight primary glioblastoma cultures that we have established. This aberrant localization reduced gap junctionnal intercellular communication by 50 to 75% as compared with primary cell cultures displaying gap junctional plaques. However, the bystander effect evaluated after lentiviral delivery of the herpes simplex virus thymidine kinase gene and ganciclovir treatment was detected in all Cx43-positive primary cell cultures, and it was independant of the Cx43 localization. These findings may have important clinical implications for the design of anticancer cytotoxic therapies that rely on the gap junction-mediated bystander effect for their success.

Rapid Changes in Connexin-43 in Response to Genotoxic Stress Stabilize Cell–Cell Communication in Corneal Endothelium

To determine how corneal endothelial (CE) cells respond to acute genotoxic stress through changes in connexin-43 (Cx43) and gap junction intercellular communication (GJIC). Cultured bovine CE cells were exposed to mitomycin C or other DNA-damaging agents. Changes in the levels, stability, binding partners, and trafficking of Cx43 were assessed by Western blot analysis and immunostaining. Live-cell imaging of a Cx43-green fluorescent protein (GFP) fusion protein was used to evaluate internalization of cell surface Cx43. Dye transfer and fluorescent recovery after photobleaching (FRAP) assessed GJIC. After genotoxic stress, Cx43 accumulated in large gap junction plaques, had reduced zonula occludens-1 binding, and displayed increased stability. Live-cell imaging of Cx43-GFP plaques in stressed CE cells revealed reduced gap junction internalization and degradation compared to control cells. Mitomycin C enhanced transport of Cx43 from the endoplasmic reticulum to the cell surface and formation of gap junction plaques. Mitomycin C treatment also protected GJIC from disruption after cytokine treatment. These results show a novel CE cell response to genotoxic stress mediated by marked and rapid changes in Cx43 and GJIC. This stabilization of cell-cell communication may be an important early adaptation to acute stressors encountered by CE.

Connexin26 expression in brain parenchymal cells demonstrated by targeted connexin ablation in transgenic mice

Astrocytes are known to express the gap junction forming proteins connexin30 (Cx30) and connexin43 (Cx43), but it has remained controversial whether these cells also express connexin26 (Cx26). To investigate this issue further, we examined immunofluorescence labelling of glial connexins in wild-type vs. transgenic mice with targeted deletion of Cx26 in neuronal and glial cells (Cx26fl/fl:Nestin-Cre mice). The Cx26 antibodies utilized specifically recognized Cx26 and lacked cross reaction with highly homologous Cx30, as demonstrated by immunoblotting and immunofluorescence in Cx26-transfected and Cx30-transfected C6 glioma cells. Punctate immunolabelling of Cx26 with these antibodies was observed in leptomeninges and subcortical brain regions. This labelling was absent in subcortical areas of Cx26fl/fl:Nestin-Cre mice, but persisted in leptomeningeal tissues of these mice, thereby distinguishing localization of Cx26 between parenchymal and non-parenchymal tissue. In subcortical brain parenchyma, Cx26-positive puncta were often co-localized with astrocytic Cx43, and some were localized along astrocyte cell bodies and processes immunolabelled for glial fibrillary acidic protein. Cx26-positive puncta were also co-localized with punctate labelling of Cx47 around oligodendrocyte somata. Comparisons of Cx26 labelling in rodent species revealed a lower density of Cx26-positive puncta and a more restricted distribution in subcortical regions of mouse compared with rat brain, perhaps partly explaining reported difficulties in detection of Cx26 in mouse brain parenchyma using antibodies or Cx26 gene reporters. These results support our earlier observations of Cx26 expression in astrocytes and its ultrastructural localization in individual gap junction plaques formed between astrocytes as well as in heterotypic gap junctions between astrocytes and oligodendrocytes.

Detailed regulatory mechanism of the interaction between ZO-1 PDZ2 and connexin43 revealed by MD simulations.

The gap junction protein connexin43 (Cx43) binds to the second PDZ domain of Zonula occludens-1 (ZO-1) through its C-terminal tail, mediating the regulation of gap junction plaque size and dynamics. Biochemical study demonstrated that the very C-terminal 12 residues of Cx43 are necessary and sufficient for ZO-1 PDZ2 binding and phosphorylation at residues Ser (-9) and Ser (-10) of the peptide can disrupt the association. However, only a crystal structure of ZO-1 PDZ2 in complex with a shorter 9 aa peptide of connexin43 was solved experimentally. Here, the interactions between ZO-1 PDZ2 and the short, long and phosphorylated Cx43 peptides were studied using molecular dynamics (MD) simulations and free energy calculation. The short peptide bound to PDZ2 exhibits large structural variations, while the extension of three upstream residues stabilizes the peptide conformation and enhanced the interaction. Phosphorylation at Ser(-9) significantly weakens the binding and results in conformational flexibility of the peptide. Glu210 of ZO-1 PDZ2 was found to be a key regulatory point in Cx43 binding and phosphorylation induced dissociation.

Atomic Force Microscopy of Connexin40 Gap Junction Hemichannels Reveals Calcium-dependent Three-dimensional Molecular Topography and Open-Closed Conformations of Both the Extracellular and Cytoplasmic Faces

Atomic force microscopy was used to study the three-dimensional molecular topography and calcium-sensitive conformational changes of Connexin40 hemichannels (connexons) reconstituted in 1,2-dioeloyl-sn-glycero-3-phosphatidylcholine lipid bilayers. Two classes of objects were observed that differed in their protrusion heights above the bilayer (2.6 versus 4.2 nm). Comparison to reconstituted connexons containing Connexin40 truncated to eliminate most of its C-terminal cytoplasmic domain showed that the two height classes corresponded to the shorter extracellular and taller cytoplasmic aspects of the hemichannels and that the C-terminal tail of Connexin40 contributes ∼1.6 nm in thickness. Hemichannels imaged in solutions containing < 10 μm Ca(2+) showed 3.1-3.2 nm depressions (openings) in 30% of the cytoplasmic faces and 65% of the extracellular faces, and high-resolution three-dimensional topography of extracellular or cytoplasmic aspects of some connexons was observed. After addition of 3.6 mm Ca(2+), > 75% of the connexons in either orientation adopted closed conformations. In contrast, hemichannels imaged in the presence of 0.1 mm EDTA showed large (5.6- to 5.8-nm diameter) openings in nearly all hemichannels regardless of orientation, and detailed topography was visible in many connexons. Real-time imaging following the addition of 3.6 mm Ca(2+) showed transitions of both extracellular and cytoplasmic orientations from "open" into "closed" conformations within several minutes. These studies provide the first high-resolution topographic information regarding a connexin with a large cytoplasmic domain and suggest that the extramembranous portions of Connexin40 contribute to a channel entrance that is relaxed by chelation of residual divalent cations.

Asparagine 175 of Connexin32 Is a Critical Residue for Docking and Forming Functional Heterotypic Gap Junction Channels with Connexin26

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.

Ocular Pathology Relevant to Glaucoma in aGja1Jrt/+Mouse Model of Human Oculodentodigital Dysplasia

Oculodentodigital dysplasia (ODDD) is a human disorder caused by mutations in the gap junction alpha 1 (GJA1) gene encoding the connexin43 (Cx43) gap junction protein. Causal links between GJA1 mutations and glaucoma are not understood. The purpose in this study was to examine the ocular phenotype for Gja1(Jrt/+) mice harboring a Cx43 G60S mutation. METHODS; In young Gja1(Jrt/+) mice, Cx43 abundance was assessed with a Western blot, and Cx43 localization was visualized using immunohistochemistry and confocal microscopy. Intraocular pressure (IOP) was measured by rebound tonometry, and eye anatomy was imaged using ocular coherence tomography (OCT). Hematoxylin and eosin (H&E)-stained eye sections were examined for ocular histopathology related to the development of glaucoma. Decreased Cx43 protein levels were evident in whole eyes from Gja1(Jrt/+) mice compared with those of wild-type mice at postnatal day 1 (P = 0.005). Cx43 immunofluorescence in ciliary bodies of Gja1(Jrt/+) mice was diffuse and intracellular, unlike the gap junction plaques prevalent in wild-type mice. IOP in Gja1(Jrt/+) mice changed during postnatal development, with significantly lower IOP at 21 weeks of age in comparison to the IOP of wild-type eyes. Microphthalmia, enophthalmia, anterior angle closure, and reduced pupil diameter were observed in Gja1(Jrt/+) mice at all ages examined. Ocular histology showed prominent separations between the pigmented and nonpigmented ciliary epithelium of Gja1(Jrt/+) mice, split irides, and alterations in the number and distribution of nuclei in the retina. Detailed phenotyping of Gja1(Jrt/+) eyes offers a framework for elucidating human ODDD ocular disease mechanisms and evaluating new treatments designed to protect ocular synaptic network integrity.

Phosphorylation of Connexin 50 by Protein Kinase A Enhances Gap Junction and Hemichannel Function

Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ∼200-pS open state while simultaneously reducing transitions between this open state and a ∼65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ∼200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers.

Ca2+ homeostasis defects and hereditary hearing loss

Ca(2+) acts as a fundamental signal transduction element in inner ear, delivering information about sound, acceleration and gravity through a small number of mechanotransduction channels in the hair cell stereocilia and voltage activated Ca(2+) channels at the ribbon synapse, where it drives neurotransmission. The mechanotransduction process relies on the endocochlear potential, an electrical potential difference between endolymph and perilymph, the two fluids bathing respectively the apical and basolateral membrane of the cells in the organ of Corti. In mouse models, deafness and lack or reduction of the endocochlear potential correlate with ablation of connexin (Cx) 26 or 30. These Cxs form heteromeric channels assembled in a network of gap junction plaques connecting the supporting and epithelial cells of the organ of Corti presumably for K(+) recycle and transfer of key metabolites, for example, the Ca(2+) -mobilizing second messenger IP(3) . Ca(2+) signaling in these cells could play a crucial role in regulating Cx expression and function. Another district where Ca(2+) signaling alterations link to hearing loss is hair cell apex, where ablation or missense mutations of the PMCA2 Ca(2+) -pump of the stereocilia cause deafness and loss of balance. If less Ca(2+) is exported from the stereocilia, as in the PMCA2 mouse mutants, Ca(2+) concentration in endolymph is expected to fall causing an alteration of the mechanotransduction process. This may provide a clue as to why, in some cases, PMCA2 mutations potentiated the deafness phenotype induced by coexisting mutations of cadherin-23 (Usher syndrome type 1D), a single pass membrane Ca(2+) binding protein that is abundantly expressed in the stereocilia.

The large GTPase dynamin2: A new player in connexin 43 gap junction endocytosis, recycling and degradation

Connexins (Cx) are key regulators of cell proliferation, differentiation and apoptosis. Cx trafficking and endocytosis need interactions with a large number of signaling and scaffolding proteins. We demonstrate herein that Cx43-GFP gap junction plaque endocytosis was blocked in cells transfected by the dominant-negative form of dynamin2 (Dyn2K44A) and by dynasore, an inhibitor of dynamin GTPase activity, which reduced the association between dynamin2 and Cx43. Our data also reveal that recruitment of the GTPase at the plasma membrane and its activation by c-Src are key events for Cx43 internalization. In addition they show that dynamin2 participated in internalization and degradation of the gap junction plaque but also in recycling of Cx43 to the plasma membrane through respectively Rab5/Rab7 and Rab11 pathways. These results demonstrate for the first time that dynamin2 is a new Cx partner and report an innovating mechanistic model by which dynamin2 may control Cx43 gap junction plaque invagination, endocytosis, recycling and degradation. These processes are magnified in response to carcinogen exposure underlining their potential importance during carcinogenesis.

Development of gap junctional intercellular communication within the lateral wall of the rat cochlea

Auditory function depends on gap junctional intercellular communication (GJIC) between fibrocytes within the cochlear spiral ligament, and basal cells and intermediate cells within stria vascularis. This communication within the lateral wall is hypothesized to support recirculation of K + from perilymph to the intra-strial space, and thus is essential for the high [K +] measured within endolymph, and the generation of the endocochlear potential. In rats, the [K +] within endolymph reaches adult levels by postnatal day 7 (P7), several days before hearing onset, suggesting that GJIC matures before auditory responses are detectable. In this study we have mapped the postnatal development of GJIC within the cochlear lateral wall, to determine the stage at which direct communication first exists between the spiral ligament and stria vascularis. Connexin 30 immunofluorescence revealed a progressive increase of gap junction plaque numbers from P0 onwards, initially in the condensing mesenchyme behind strial marginal cells, and spreading throughout the lateral wall by P7–P8. Whole-cell patch clamp experiments revealed compartmentalized intercellular dye-coupling in the lateral wall between P2 and P5. There was extensive dye-coupling throughout the fibrocyte syncytium by P7. Also, by P7 dye introduced to fibrocytes could also be detected within strial basal cells and intermediate cells. These data suggest that lateral wall function matures several days in advance of hearing onset, and provide anatomical evidence of the existence of a putative K + recirculation pathway within the cochlear lateral wall.

Single Hemichannels Recorded in Lipid Bilayers and Artificial Gap Junction Formation with Cells

Connexins (Cx) are members of a multigene family of membrane-spanning proteins that form gap junctions, which are composed of two hexameric hemichannels, called connexons. These gap junctions, organized in so-called gap junctional plaques, span the extracellular space/matrix of adjacent cells and thus allow a passive exchange of small molecules up to about 1 kDa. Connexins are widely distributed with various subtypes of connexin and are involved in different biological processes such transmission of information and propagation of action potential for e.g. Recent studies indicates that hemichannels do open under physiological and pathological conditions. In our study, we investigated the biophysical properties of hemichannels Cx26 and Cx43 which were isolated biochemically and reconstituted into synthetic lipid membranes. Both hemichannels are present in different tissues and involved in different pathologies. The results on a study of the Cx26 are presented. Reconstitutions of functional Cx26 and mutant hemichannels were performed. Secondly, Cx43 was purified and reconstituted into bilayers. The hemichannel Cx43 properties were compared to previous studies and showed similarities of conductance on single channel recordings of Cx43 in cells. Our focus was then to form artificial gap junctions, first between two unrelated cells and then between cells and bilayers containing functional hemichannels. This was done using Cx26 or Cx43. The bilayer-cell configuration allows to measure electrophysiological properties of the cells indirectly via gap junctions. Single channel recordings of gap junctions were recorded using a bilayer containing Cx43 and Cardiomyocytes expressing Cx43. Macroscopic currents were as well recorded between bilayers and cell lines expressing Cx26 or Cx43.

Binding Kinetics of Inter-Connexon Interaction

Gap junctions are pairs of hexameric half-channels called connexons, which coaxially dock to connect two adjacent cells, mediating both adhesion and channeling between cells in many types of tissue. Connexons are formed of six connexin proteins (Cx). Gap junctions form by interdigitating the two extracellular loops of each connexin. While gap junction structure and function has been widely characterized using different techniques, the binding affinity of the inter-connexon interaction remains unknown. The goal of this work was to determine the binding affinity of gap junctions using dynamic force spectroscopy atomic force microscopy (AFM). Among the residues that mediate inter-connexon interaction, an exposed stretch of conserved amino acids ‘NTVD’ within the extracellular loop 2 (E2) has been identified. For dynamic force spectroscopy, we covalently linked mimetic peptides ‘NTVD‘ that mimic loop E2 of Cx26 to the AFM tip, while Cx26 two-dimensional (2D) crystals were immobilized on a mica substrate. We report the first characterization of the binding strength of the gap junction interaction. Force curves at various retraction speeds were acquired to determine the dissociation kinetics of the peptide-Cx26 interaction, while adhesion probability measurements at different contact times revealed the binding kinetics. The relatively fast intrinsic dissociation rate (koff) inferred a rather dynamic inter-connexon interaction, while the slow association rate (kon) probably reflects the restricted mobility and degrees of freedom of the connexons in the densely packed organization observed in native gap junction plaques and the reduced flexibility and dimensions of the extracellular loops. Our results suggest that gap junction formation may occur before plaque formation.

Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50.

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.

A dominant connexin43 mutant does not have dominant effects on gap junction coupling in astrocytes

Dominant mutations in GJA1, the gene encoding the gap junction protein connexin43 (Cx43), cause oculodentodigital dysplasia (ODDD), a syndrome affecting multiple tissues, including the central nervous system (CNS). We investigated the effects of the G60S mutant, which causes a similar, dominant phenotype in mice (Gja1(Jrt/+)). Astrocytes in acute brain slices from Gja1(Jrt/+) mice transfer sulforhodamine-B comparably to that in their wild-type (WT) littermates. Further, astrocytes and cardiomyocytes cultured from Gja1(Jrt/+) mice showed a comparable transfer of lucifer yellow to those from WT mice. In transfected cells, the G60S mutant formed gap junction (GJ) plaques but not functional channels. In co-transfected cells, the G60S mutant co-immunoprecipitated with WT Cx43, but did not diminish GJ coupling as measured by dual patch clamp. Thus, whereas G60S has dominant effects, it did not appreciably reduce GJ coupling.

Impaired membrane targeting and aberrant cellular localization of human Cx26 mutants associated with inherited recessive hearing loss

Conclusion: This study demonstrated that five Cx26 mutations (R32H, S199F, 572delT, 631-632delGT, and Y155X) affect gap junction (GJ) functions by causing impaired membrane targeting and aberrant cellular localization, and one mutation (R165W) leads to a constriction of the channel pore with no dye coupling. Objective: To investigate the pathogenetic roles of six recessive Cx26 mutations (p.R32H, p.R165W, p.S199F, c.572delT, c.631-632delGT, and p.Y155X), which have not been functionally analyzed in vitro. Methods: The six mutants and wild-type Cx26 (wtCx26) were cloned into the EcoRI and SalI sites of pEGFP-N1 vector. We transfected the seven constructs into HeLa cells, followed by analysis of their protein expression using the western blot method, study of the protein localizations and gap junction-plaques on the cytomembrane under confocal microscopy, and assessment of the dye coupling of the mutated GJ channels by intercellular dye transfer experiment. Results: p.R165W targeted the cytomembrane and formed GJ-like structures in adjacent HeLa cells, causing null dye coupling. The mutants (p.R32H, p.S199F, c.572delT, c.631-632delGT, and p.Y155X) failed to reach the cell surface, and perfectly co-localized with endoplasmic reticulum (ER) throughout the cells.

Marine sponge depsipeptide increases gap junction length in HTC cells transfected with Cx43–GFP

Connexins are membrane proteins that form GJ (gap junction) channels between adjacent cells. Cx43 (connexin 43), the most widely expressed member of the connexin family, has a rapid turnover rate, and its degradation involves both the lysosomal and ubiquitin–proteasome pathway. The goal of this work was to study the effects of geodiamolides, natural peptides from marine sponge that normally are involved with microfilament disruption, on connexin assembly or degradation in the plasma membrane. HTC (hepatocarcinoma cells) expressing Cx43–GFP (green fluorescent protein) were submitted to treatment with 200 nM geodiamolides A, B, H and I for 2 and 4 h. Microfilament distribution and the presence and size of GJ plaques were evaluated by laser scanning confocal microscopy. Among the four peptides tested, only Geo H (geodiamolide H) statistically enhanced the length of GJ plaques. Geodiamolide A also showed activity in the GJ plaque size; however, its effect was less pronounced. Treatment with Geo H could interfere with the delivery of connexins to the degradation structures, similar to proteasomal pathways, keeping the connexins assembled and accumulating GJ plaques. Further experiments, with the cells treated with Geo H, using the fungal antibiotic BFA (brefeldin A), were performed in order to uncouple events leading to GJ assembly from those related to GJ removal, since BFA is known to block protein trafficking within a fused ER (endoplasmic reticulum)/Golgi compartment. GJ plaques were drastically reduced after BFA/Geo H treatment, thus indicating that Geo H affects mainly the delivery pathway of Cx43 protein.