Assembly of heteromeric connexons in guinea-pig liver en route to the Golgi apparatus, plasma membrane and gap junctions.

Guinea-pig liver gap junctions are constructed from approximately equal amounts of connexins 26 and 32. The assembly of these connexins into connexon hemichannels and gap junctions was studied using antibodies specific to each connexin. Intracellular membranes were shown to contain low amounts of connexin 26 relative to connexin 32 in contrast to the equal connexin ratios detected in lateral plasma membranes and gap junctions. Assembly of gap junctions requires oligomerization of connexins into connexons that may be homomeric or heteromeric. Immunoprecipitation using antibodies to connexins 26 and 32 showed that liver gap junctions were heteromeric. A chemical cross-linking procedure showed that connexons solubilized from guinea-pig liver gap junctions were constructed of hexameric assemblies of connexin subunits. The intracellular site of oligomerization of connexins was investigated by velocity sedimentation in sucrose-detergent gradients. Oligomers of connexins 26 and 32 were extensively present in Golgi membranes and oligomeric intermediates, especially of connexin 26, were detected in the endoplasmic reticulum-Golgi intermediate subcellular fraction. Two intracellular trafficking pathways that may account for the delivery of connexin 26 to the plasma membrane and explain the heteromeric nature of liver gap junctions are discussed.     

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.     

Maize mesocotyl plasmodesmata proteins cross-react with connexin gap junction protein antibodies.

Polypeptide present in various cell fractions obtained from homogenized maize mesocotyls were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotted, and screened for cross-reactivity with antibodies against three synthetic polypeptides spanning different regions of the rat heart gap junctional protein connexin43 and the whole mouse liver gap junctional protein connexin32. An antibody raised against a cytoplasmic loop region of connexin43 cross-reacted strongly with a cell wall-associated polypeptide (possibly a doublet) of 26 kilodaltons. Indirect immunogold labeling of thin sections of mesocotyl tissue with this antibody labeled the plasmodesmata of cortical cells along the entire length of the plasmodesmata, including the neck region and the cytoplasmic annulus. Sections labeled with control preimmune serum were essentially free of colloidal gold. An antibody against connexin32 cross-reacted with a 27-kilodalton polypeptide that was present in the cell wall and membrane fractions. Indirect immunogold labeling of thin sections with this antibody labeled the plasmodesmata mainly in the neck region. It is suggested that maize mesocotyl plasmodesmata contain at least two different proteins that have homologous domains with connexin proteins.     

Specific localization of gap junction protein, connexin45, in the deep muscular plexus of dog and rat small intestine

Cellular networks of pacemaker activity in intestinal movements are still a matter of debate. Because gap-junctional intercellular communication in the intestinal wall may provide important clues for understanding regulatory mechanisms of intestinal movements, we have attempted to clarify the distribution patterns of three types of gap junction proteins. Using antibodies for connexin40, connexin43, connexin45, smooth muscle actin, and vimentin, immunocytochemical observations were made with the confocal laser scanning microscope on cryosections of fresh-frozen small intestine and colon of the dog and rat. Connexin 45 was localized along the deep muscular plexus of the small intestine in both dog and rat. Double labeling studies revealed that connexin45 overlapped with vimentin –, but not actin-positive areas, indicating the fibroblast-like nature of the cells, rather than their being smooth muscle-like. Connexin43 immunoreactivity appeared along the smooth muscle cell surface in the outer circular layer of the small intestine of both animals. Connexin 40 immunoreactivity was not observed in the muscle layer other than in the wall of large blood vessels. It is suggested that connexin45-expressing cells along the deep muscular plexus of dog and rat small intestine are likely to act as a constituent of a pacemaker system, which may include a conductive system, by forming a cellular network operating via specific types of gap junctions.     

Immunochemical characterization of the gap junction protein connexin45 in mouse kidney and transfected human HeLa cells

Antibodies to the gap junction protein connexin45 (Cx45) were obtained by immunizing rabbits with fusion protein consisting of glutathione S-transferase and 138 carboxy-terminal amino acids of mouse Cx45. As shown by immunoblotting and immunofluorescence, the affinity-purified antibodies recognized Cx45 protein in transfected human HeLa cells as well as in the kidney-derived human and hamster cell lines 293 and BHK21, respectively. In Cx45-transfected HeLa cells, this protein is phosphorylated as demonstrated by immunoprecipitation after metabolic labeling. The phosphate label could be removed by treatment with alkaline phosphatase. A weak phosphorylation of Cx45 protein was also detected in the cell lines 293 and BHK21. Treatment with dibutyryl cyclic adenosine or guanosine monophosphate (cAMP, cGMP) did not alter the level of Cx45 phosphorylation, in either Cx45 transfectants or in 293 or BHK21 cells. The addition of the tumor-promoting agent phorbol 12-myristate 13-acetate (TPA) led to an increased ³²P phosphate incorporation into the Cx45 protein in transfected cells.The Cx45 protein was found in homogenates of embryonic brain, kidney, and skin, as well as of adult lung. In kidney of four-day-old mice, Cx45 was detected in glomeruli and distal tubules, whereas connexin32 and –26 were coexpressed in proximal tubules. No connexin43 protein was detected in renal tubules and glomeruli at this stage of development. Our results suggest that cells in proximal and distal tubules are interconnected by gap junction channels made of different connexin proteins. The Cx45 antibodies characterized in this paper should be useful for investigations of Cx45 in renal gap junctional communication.     

Ilimaquinone inhibits gap junctional communication in a connexin isotype-specific manner

Ilimaquinone (IQ) and brefeldin A (BFA) disrupt the Golgi complex structure and block protein transport to the plasma membrane, and inhibit gap junctional communication. HeLa cells expressing rat connexin26, 32, or 43, or mouse connexin31, 36, 45, or 57, were used to study the response patterns of gap junctional communication (dye transfer) to ilimaquinone, brefeldin, and the potent protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA). 12-O-Tetradecanoylphorbol-13-acetate (followed for 2 h) caused dose- and time-dependent decreases in communication for five of seven connexins, the unresponsive being connexin45 and 57. Brefeldin (followed for 6 h) caused dose- and time-dependent decreases in communication for six of seven connexins, the exception being connexin26. These results are consistent with Golgi-mediated transport to the cell membrane for all connexins except connexin26. In contrast, ilimaquinone (followed for 6 h) caused a rapid (15-30 min) and nearly complete inhibition of dye transfer through connexin43 channels. For the other connexins, there was a slow and weak response for connexin26, 31, and 32, reaching 65-70% of control communication level, while connexin36, 45, and 57 were unresponsive. Thus, among the tested connexins, ilimaquinone has a strong specificity for connexin43, and the mechanism appears independent of the Golgi complex and of protein kinase C.     

Degradation of connexins from the plasma membrane is regulated by inhibitors of protein synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Degradation of Connexins from the Plasma Membrane Is Regulated by Inhibitors of Protein Synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Proteomic analysis of blastema formation in regenerating axolotl limbs

Background

For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood.

Results

Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred.

Conclusion

This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.     

Expression and functions of neuronal gap junctions

Gap junctions are channel-forming structures in contacting plasma membranes that allow direct metabolic and electrical communication between almost all cell types in the mammalian brain. At least 20 connexin genes and 3 pannexin genes probably code for gap junction proteins in mice and humans. Gap junctions between murine neurons (also known as electrical synapses) can be composed of connexin 36, connexin 45 or connexin 57 proteins, depending on the type of neuron. Furthermore, pannexin 1 and 2 are likely to form electrical synapses. Here, we discuss the roles of connexin and pannexin genes in the formation of neuronal gap junctions, and evaluate recent functional analyses of electrical synapses that became possible through the characterization of mouse mutants that show targeted defects in connexin genes.     

Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis

Direct immunogold labeling and histological mapping of membrane proteins is demonstrated in Lexan-stabilized SDS-washed freeze-fracture replicas of complex tissues. Using rat brain and spinal cord as primary model systems and liver as a "control" tissue to identify preparation and labeling artifacts, we demonstrate the presence of connexin43 in freeze-fractured gap junctions of identified and mapped astrocytes and ependymocytes, and confirm the presence of connexin32 in freeze-fractured gap junctions in liver. In addition, the simultaneous double-labeling of dissimilar proteins (connexin43 and aquaporin-4) is demonstrated in gap junctions and square arrays, respectively, in the plasma membranes of astrocytes and ependymocytes. Finally, double-side shadowing and conventional staining methods are used to reveal the extent of biological material present at the time of labeling and to investigate the dynamics of membrane solubilization, the primary artifacts that occur during labeling, and several factors limiting quantitative analysis.     

Characterization of the gap junction protein,connexin45

Connexin45 is a gap junction protein which forms channels with unique characteristics. RNA blots demonstrated that connexin45 is expressed in a number of cell lines including WB, SK Hepl, BHK, A7r5, CLEM, and BWEM cells. Connexin45 was further studied in BWEM cells using specific affinity-purified antibodies directed against a synthetic peptide representing amino acids 285–298 of its sequence. Immunofluorescence experiments demonstrated that the BWEM cells expressed both connexin43 and connexin45 and that these connexins colocalized. Connexin45 polypeptide, immunoprecipitated from BWEM cells metabolically labeled with [³⁵S]-methionine, consisted of a predominant 48 kD polypeptide. Connexin45 and connexin43 contained radioactive phosphate when immunoprecipitated from BWEM cells metabolically labeled with [³²P]-orthophosphoric acid. This phosphate label was removed from connexin45 by alkaline phosphatase digestion. Treatment of BWEM cells with the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited intercellular passage of microinjected Lucifer yellow. While TPA treatment induced phosphorylation of connexin43 in these cells, it reduced the expression of connexin45. Furthermore, the connexin45 expressed after TPA treatment was not phosphorylated. These results suggest that treatments which alter protein phosphorylation may regulate connexin43 and connexin45 in BWEM cells by different mechanisms.These studies were supported by National Institutes of Health grants HL45466 and EY08368. J.G.L. is supported by a fellowship from the Lucille P. Markey Foundation. E.C.B. is an Established Investigator of the American Heart Association.     

Poly(ADP-ribose) polymerase cleavage during apoptosis: when and where?

On freeze-fracture replicas, gap junctions are frequently colocalized with tight junctions. In this study, to elucidate the relationship between gap- and tight-junction proteins, we investigated the localization of gap-junction proteins Cx32 and Cx26 and tight-junction proteins occludin, claudin-1, ZO-1, and ZO-2 in primary cultured rat hepatocytes, using confocal laser microscopy. In hepatocytes cultured in 2% DMSO and 10(-7) M glucagon medium, Cx32- but not Cx26-immunoreactive lines were observed on the most subapical plasma membrane at cell borders, while on the basolateral membrane both Cx32- and Cx26-positive spots were colocalized. Occludin-, claudin-1-, ZO-1-, and ZO-2-immunoreactive lines were also linearly observed on the most subapical plasma membrane and were colocalized with only Cx32-immunoreactive lines. In freeze-fracture analysis, many small gap-junction plaques were observed within a well-developed tight-junction strand network. The fence function of tight junctions in the cells, as examined by diffusion of labeled sphingomyelin, was well maintained. We also carried out Western blotting for Cx32 following immunoprecipitation with anti-occludin, anti-claudin-1, or anti-ZO-1 antibodies. Cx32 was detectable in all immunoprecipitates. These results suggest that Cx32 gap junctions, but not those with Cx26, are closely coordinated with the expression and function of tight junctions in hepatocytes and that Cx32 gap-junction formation may affect cell polarity through modification of tight-junction expression.     

Gap junctions: structure and function (Review)

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to ~1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.     

Expression of gap junction genes during postnatal neural development

The timing of appearance of mRNAs encoding gap junction proteins was examined during development of the rat and mouse brain. Complementary DNAs (cDNAs) specific for the mRNA for the liver-type gap junction protein, connexin32, and the heart-type gap junction protein, connexin43, were used to probe Northern blots of total RNA isolated from the forebrain and hindbrain of mice and rats at various times before and after birth. Prior to postnatal day 10, connexin32 mRNA is detectable only at low levels. By postnatal days 10 to 16, a sharp increase occurs in the level of this mRNA. This increase is detectable first in the hindbrain, and subsequently in the forebrain. In contrast, connexin43 mRNA is readily detectable at birth, and the level of this mRNA also increases during subsequent development. The developmental appearance of the gap junction proteins, connexin32 and connexin43, was similar to that of their respective mRNAs. These results indicate that the genes encoding connexin32 and connexin43 are differentially expressed during neural development.     

Expression of gap junction genes during postnatal neural development.

The timing of appearance of mRNAs encoding gap junction proteins was examined during development of the rat and mouse brain. Complementary DNAs (cDNAs) specific for the mRNA for the liver-type gap junction protein, connexin32, and the heart-type gap junction protein, connexin43, were used to probe Northern blots of total RNA isolated from the forebrain and hindbrain of mice and rats at various times before and after birth. Prior to postnatal day 10, connexin32 mRNA is detectable only at low levels. By postnatal days 10 to 16, a sharp increase occurs in the level of this mRNA. This increase is detectable first in the hindbrain, and subsequently in the forebrain. In contrast, connexin43 mRNA is readily detectable at birth, and the level of this mRNA also increases during subsequent development. The developmental appearance of the gap junction proteins, connexin32 and connexin43, was similar to that of their respective mRNAs. These results indicate that the genes encoding connexin32 and connexin43 are differentially expressed during neural development.     

Changes in Cellular Distribution of Connexins 32 and 26 during Formation of Gap Junctions in Primary Cultures of Rat Hepatocytes

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sectionsin vivo.Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.     

Low molecular weight GTP-binding proteins in cardiac muscle. Association with a 32-kDa component related to connexins.

Low molecular weight GTP-binding proteins and their cellular interactions were examined in cardiac muscle. Heart homogenate was separated into various subcellular fractions by differential and sucrose density gradient centrifugation. Various fractions were separated by sodium dodecyl sulfate-gel electrophoresis, blotted to nitrocellulose, and GTP-binding proteins detected by incubating with [alpha-32]GTP. Three polypeptides of M(r) 23,000, 26,000, and 29,000 were specifically labeled with [alpha-32P]GTP in all the fractions examined and enriched in sarcolemmal membranes. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 26- and 29-kDa polypeptides. A polypeptide of M(r) 40,000 was weakly labeled with [alpha-32P]GTP in the sarcolemmal membrane and tentatively identified as Gi alpha by immunostaining with anti-Gi alpha antibodies. Cytosolic GTP-binding proteins were labeled with [alpha-32P]GTP and their potential sites of interaction investigated using the blot overlay approach. A polypeptide of 32 kDa present in sarcolemmal membranes, intercalated discs, and enriched in heart gap junctions was identified as a major site of interaction. The low molecular weight GTP-binding proteins associated with the 32-kDa polypeptide through a complex involving cytosolic components of M(r) 56,000, 36,000, 26,000, 23,000, and 12,000. A monoclonal antibody against connexin 32 from liver strongly recognized the 32-kDa polypeptide in heart gap junctions, whereas polyclonal antibodies only weakly reacted with this polypeptide. The low molecular weight GTP-binding proteins associated with a 32-kDa polypeptide in liver membranes that was also immunologically related to connexin 32. These results indicate the presence of a subset of low molecular weight GTP-binding proteins in a membrane-associated and a cytoplasmic pool in cardiac muscle. Their association with a 32-kDa component that is related to the connexins suggests that these polypeptides may be uniquely situated to modulate communication at the cell membrane.