Molecular organization of gap junction membrane channels

Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas formingin vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.     

Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

To examine the role of gap junctions in cell senescence, the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore, cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis, p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin(10mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis, elevation of p53 expression, loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.     

Preendocytotic alterations in cumulus cell gap junctions precede meiotic resumption in the rat cumulus-oocyte complex

Cumulus cells in the mammalian ovary are normally connected to each other and to their enclosed oocyte by an extensive network of gap junctions (GJs). We have shown that the loss of cumulus cell GJs is correlated temporally with meiotic resumption in the intact preovulatory rat follicle (Larsen et al., 1986). Here we describe morphological changes in GJ particle packing patterns (PPPs) that occur prior to GJ loss and meiotic resumption in hormonally stimulated rat cumulus-oocyte complexes (COCs). In the PMSG-primed rat, 89% of the cumulus cell GJ area detected by freeze-fracture electron microscopy consists of tightly packed junctional particles: 4% exhibit loose PPPs of randomly dispersed particles; and 7% contain a mixture of both tight and loose PPPs. One to 2 hr after stimulation with hCG, the area of GJs containing tight PPPs drops by 50%-60%, while junctions exhibiting loosely organized and mixed patterns increase concomitantly. These shifts in PPPs are accompanied by the appearance of unusual particle-free areas of puckered or ruffled nonjunctional membrane at the GJ periphery. Cumulus cell GJs from isolated COCs incubated in FSH-containing medium demonstrate a similar shift in PPPs prior to meiotic resumption. The appearance of fusing areas of particle-free nonjunctional membrane at the GJ periphery in vitro is correlated with GJ loss and is not seen in COCs treated with dihydrocytochalasin B to inhibit endocytotic removal of cumulus GJs. The structural and temporal nature of these morphological observations supports the hypothesis that interruption of junctional communication plays a role in meiotic maturation of the preovulatory oocyte.     

ELF magnetic field inhibits gap junctional intercellular communication and induces hyperphosphorylation of connexin43 in NIH3T3 cells.

The effects of extremely low frequency (ELF) magnetic field on gap junctional intercellular communication (GJIC), protein levels, and phosphorylation of connexin43 (Cx43) were studied in NIH3T3 cells. The suppression of GJIC by 24 h, 50 Hz, 0.8 mT ELF magnetic field, 2 h, 3 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), or ELF combined with TPA treatment was confirmed by the fluorescence recovery after photobleaching (FRAP) analysis with a confocal microscope. The results showed that ELF or TPA exposure induced 50-60% inhibition of GJIC (P < 0.01). ELF combined with TPA enhanced the inhibition of GJIC. Western blot analysis using Cx43 specific antibodies showed obviously decreasing non phosphorylated Cx43 (P(0)) induced by ELF and/or TPA exposure. On the other hand, cells treated with ELF and/or TPA displayed a hyperphosphorylated Cx43 band (P(3)). However, there was no obvious changes in the level of Cx43 protein. The results implied that the P(3) band appeared to result from phosphorylation of P(0). But it remains possible that upon the ELF exposure P(0) is converted to P(1), P(2) or both and that P(3) is formed from P(1) or P(2) resulting in the observed hyperphosphorylation pattern. From the present study, we conclude that ELF magnetic field inhibits GJIC and the main mechanism is the hyperphosphorylation of Cx43.     

Isolation and characterization of gap junctions from Drosophila melanogaster

Summary A procedure has been developed to isolate gap junction-enriched subcellular fractions from Drosophila. Crude membranes from larval homogenates were extracted with 1% N-lauroyl sarcosine in 6 M urea and the gap junctions were collected by centrifugation. The major proteins were separated by SDS PAGE and purified by electro-elution. Electron microscopy revealed structurally pleiomorphic gap junctions in the fractions which included (1) conventional, 16–18 nm-wide septalaminar, (2) collapsed, 13–15 nm-wide pentalaminar, (3) split, and (4) aggregated forms. The fractions contained five major proteins with apparent molecular weights of 18, 26, 36, 52 and 54 kD. Evidence based on (1) the degradation and aggregation behavior of the major proteins following electro-elution and reelectrophoresis, (2) immunological cross-reactivities by affinity-purified antibodies against the major proteins on immunoblots, and (3) immunofluorescent staining of presumptive gap junctions in Drosophila imaginal discs at the light-microscopic level and immunogold staining of purified gap junctions at the electron-microscopic level suggests that the major proteins are interrelated and of gap-junction origin.     

Intercellular communication via gap junctions affected by mechanical load in the bovine annulus fibrosus

Cells in the intervertebral disc, as in other connective tissues including tendon, ligament and bone, form interconnected cellular networks that are linked via functional gap junctions. These cellular networks may be necessary to affect a coordinated response to mechanical and environmental stimuli. Using confocal microscopy with fluorescence recovery after photobleaching methods, we explored the in situ strain environment of the outer annulus of an intact bovine disc and the effect of high-level flexion on gap junction signalling. The in situ strain environment in the extracellular matrix of the outer annulus under high flexion load was observed to be non-uniform with the extensive cellular processes remaining crimped sometimes at flexion angles greater than 25°. A significant transient disruption of intercellular communication via functional gap junctions was measured after 10 and 20 min under high flexion load. This study illustrates that in healthy annulus fibrosus tissue, high mechanical loads can impede the functioning of the gap junctions. Future studies will explore more complex loading conditions to determine whether losses in intercellular communication can be permanent and whether gap junctions in aged and degenerated tissues become more susceptible to load. The current research suggests that cellular structures such as gap junctions and intercellular networks, as well as other cell–cell and cell–matrix interconnections, need to be considered in computational models in order to fully understand how macroscale mechanical signals are transmitted across scales to the microscale and ultimately into a cellular biosynthetic response in collagenous tissues.     

Tetracysteine Genetic Tags Complexed with Biarsenical Ligands as a Tool for Investigating Gap Junction Structure and Dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.     

Tetracysteine Genetic Tags Complexed with Biarsenical Ligands as a Tool for Investigating Gap Junction Structure and Dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.     

Pharmacological evidence for system-dependent involvement of protein kinase C isoenzymes in phorbol ester-suppressed gap junctional communication

Several phorbol esters are potent activators of protein kinase C. They down-regulate gap junctional intercellular communication and induce phosphorylation of connexin43, but the sensitivity and extent of responses vary much between systems. We asked whether the total protein kinase C enzyme activity or the protein kinase C isoenzyme constitution was of importance for such variations. Some fibroblastic culture systems were compared. It was concluded that the total protein kinase C enzyme activity did not determine the sensitivity to phorbol esters. Furthermore, the use of isotype-specific inhibitors of protein kinase C indicated that protein kinase C alpha, delta, and epsilon may be involved to different extents in different fibroblastic systems in the response to phorbol esters.     

Development changes in regulation of embryonic chick heart gap junctions

Summary Embryonic chick myocyte pairs were isolated from ventricular tissue of 4-day, 14-day, and 18-day heart for the purpose of examining the relationship between macroscopic junctional conductance and transjunctional voltage during cardiac development. The double whole-cell patch-clamp technique was employed to directly measure junctional conductance over a transjunctional voltage range of ±100 mV. At all ages, the instantaneous junctional current (or conductance=current/voltage) varied linearly with respect to transjunctional voltage. This initial response was followed by a time- and voltage-dependent decline in junctional current to new steady-state values. For every experiment, the steady-state junctional conductance was normalized to the instantaneous value obtained at each potential and the data was pooled according to developmental age. The mean steadystate junctional conductance-voltage relationship for each age group was fit using a two-state Boltzmann distribution described previously for other voltage-dependent gap junctions. From this model, it was revealed that half-inactivation voltage for the transjunctional voltage-sensitive conductance shifted towards larger potentials by 10 mV, the equivalent gating charge increased by approximately 1 electron, and the minimal voltage-insensitive conductance exactly doubled (increased from 18 to 36%) between 4 and 18 days of development. Decay time constants were similar at all ages examined as rate increased with increasing transjunctional potential. This data provides the first direct experimental evidence for developmental changes in the regulation of intercellular communication within a given tissue. This information is consistent with the hypothesis that developmental expression of multiple gap junction proteins (connexins) may confer different regulatory mechanisms on intercellular communication pathways within a given cell or tissue.     

心肌细胞缝隙连接重塑与心律失常

缝隙连接是相邻心肌细胞间电、化学偶联的通道,亦是心室肌成为功能性合胞体的重要结构.心肌有缝隙连接蛋白(connexin,CX) 40、43与45的表达,心室肌主要表达CX43.CX43形成的缝隙连接大部分呈点状分布于闰盘部位,心肌细胞膜侧面分布极少.心肌缺血-再灌注、肥厚、衰竭、高胆同醇与糖尿病条件下,心肌细胞缝隙连接...     

Biochemical and genetic investigations on gap junctions from mammalian cells

Gap junction protein (26K) in mouse or rat liver has been studied using a rabbit antiserum directed against the sodium dodecylsulfate denatured 26K protein from mouse liver. The liver 26K protein has been localized in gap junction plaques of hepatic plasma membranes by immuno electron microscopy. Affinity purified anti-26K antiserum showed weak cross reactivity with mouse or bovine lens gap junction protein (MIP26). This result suggests some structural homology between the different gap junction proteins in liver and lens. After partial hepatectomy of young rats the liver 26K protein appears to be degraded and later resynthesized. A variant of established Chinese hamster fibroblastoid cells has been isolated and shown to be defective in metabolic cooperation via gap junctions.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions

Chinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., mec-). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The isolated Chinese hamster CI-4 cells which expressed the mec- phenotype most stringently showed the following characteristics: 1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the mec+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (mec+). Confluent CI-4 cells grown in the presence of 1 mM db-cAMP showed 9% coupled cells. 2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The mec+ parental cells showed small gap junction plaques (0.013% of the total cell surface analyzed). 3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92% positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic fibroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68%. Other cells of spontaneous mec- phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix. 4. The mec- defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of mec-, as well as mec+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the mec- defect.     

Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator-induced G-protein coupled receptor activation

During the inflammatory response, activation of G-protein coupled receptors (GPCRs) by inflammatory mediators rapidly leads to inhibition of gap junction intercellular communication (GJIC); however, the steps that lead to this inhibition are not known. Combining high-resolution fluorescence microscopy and functional assays, we found that activation of the GPCRs PAR-1 and ET_A/B by their natural inflammatory mediator agonists, thrombin and endothelin-1, resulted in rapid and acute internalization of gap junctions (GJs) that coincided with the inhibition of GJIC followed by increased vascular permeability. The endocytosis protein clathrin and the scaffold protein ZO-1 appeared to be involved in GJ internalization, and ZO-1 was partially displaced from GJs during the internalization process. These findings demonstrate that GJ internalization is an efficient mechanism for modulating GJIC in inflammatory response.     

Rat myometrial smooth muscle cells show high levels of gap junctional communication under a variety of culture conditions

Summary Gap junctional communciation was examined in rat myometrial smooth muscle cells cultured under a variety of conditions. As a functional measure of gap junctional communication, donor cells were microinjected with the fluorescent dye, Lucifer yellow, and the transfer of dye from donor cells to primary neighbor cells was monitored by fluorescence microscopy. In a myometrial smooth muscle cell line established from midgestation (Day 10) rats, high levels of dye transfer, in excess of 90%, were observed in primary cultures and at Passages 1 and 10. A slight decrease in dye transfer to 75% was observed at Passage 5. Similarly, high levels of dye transfer were observed in a smooth muscle cell line established from the myometrium of a late-gestation (Day 19) rat under subconfluent as well as confluent culture conditions. Myometrial smooth muscle cell cultures established from sexually immature 19-day-old rats also exhibited high levels of dye transfer in primary cultures and at Passage 10. Treatment of primary myometrial smooth muscle cell cultures derived from immature 19-day-old rats with 17β-estradiol (50 ng/ml) and 4-pregnen-3,20-dione (150 ng/ml) for 48 h in vitro had no significant effect on the high levels of dye transfer. Thus, extensive dye transfer was observed in the rat myometrial smooth muscle cells under all culture conditions examined, regardless of sexual maturity or gestational stage of the animal, in vitro hormone treatment, or cell density.     

The carcinogen benzo(e)pyrene is metabolized by DM15 cells without an uncoupling effect on their gap junctions

Benzo(e)pyrene (B(e)P) promotes carcinogenesis in the skin. Unlike some other promoters however, B(e)P does notproduce an uncoupling effect on gap junction permeability in DM15 transformedfibroblasts. This study demonstrates thatDM15 cells exhibit a relatively high level of B(e)P metabolism. Moreover, although pretreatment of DM15 cells with benz(a)anthracene results in an 8-fold increase of arylhydrocarbon hydroxylase activity and a 2-fold increase in the rate ofB(e)P metabolism, it did not enable B(e)P to affectLucifer Yellow transfer between DM15 cells. We conclude that neitherB(e)P nor its metabolites are capable of uncoupling gap junction permeability in DM15 cells.Abbreviations AHH aryl hydrocarbon hyroxylase - BA benz(a)anthracene - B(a)P benzo(a)pyrene - B(e)P benzo(e)pyrene - LY Lucifer Yellow - MFO mixed-function oxidases - PAH polycyclic aromatic hydrocarbons     

Isolation and protein composition of gap junctions from rabbit hearts.

We have modified a method for isolating gap-junctional membrane from mouse hearts [Kensler & Goodenough (1980) J. Cell Biol. 86, 755-764] to isolate gap junctions of comparable purity from rabbit hearts more rapidly, with better yield, and without resort to non-ionic detergents. Purification was monitored by electron microscopy of thin-sectioned membrane pellets and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gap junctions were obtained as vesicles whose mean surface area approximated that of junctions in intact myocardial cells. About 10-20% of the vesicles were ferritin-impermeable. Approx. 125 micrograms of membrane protein was obtained per 8 g of rabbit heart. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of purified gap junctions showed five major protein bands of mol.wts. 46 000, 44 000, 33 000, 30 000 and 28 500 that co-purified with the junctions. This protein composition was nearly identical with that published for gap junctions of mouse hearts, and differed markedly from the protein composition of gap junctions from non-excitable cells (lens and liver). The constancy of junctional protein composition between hearts of two different species and its non-identity with that from liver and lens suggest that, although gap-junctional structure in mammalian tissues seems to be remarkably similar by electron-microscopic techniques, junctional-channel protein composition actually varies from tissue to tissue and may be adapted to the permeability requirements of the tissue.     

Permeability and structural studies of heart cell gap junctions under normal and altered ionic conditions

Summary The permeability and ultrastructure of communicating junctions of cultured neonatal rat ventricular cells are examined under control conditions and during treatments which raise intracellular Ca²⁺. Lucifer Yellow (487 mol wt) is used to examine junctional permeability. Under normal ionic conditions dye transfer from an injected muscle cell to neighboring muscle cells occurs rapidly (in less than 6 sec) while transfer to neighboring fibroblasts occurs more slowly. Application of monensin, which results in a partial contracture with superimposed asynchrony, or A23187, which results in a partial contracture, do not inhibit the transfer of dye between the muscle cells. A23187 did result in junctional blockade between muscle cells and fibroblasts. Freeze-fractured gap junctions from control and monensin-treated cells exhibit no distinguishable differences. Center-to-center spacing was not significantly different, 9.0 nm±1.4sd versus 9.2 nm±1.3sd, respectively; and particle diameters were virtually unchanged, 8.69 nm±0.9sd versus 8.61 nm±1.07sd, respectively. These results suggest that concentrations of intracellular Ca²⁺ sufficient to support a partial contracture and asynchronous contractile activity do not result in a block of intercellular junctions in cultured myocardial cells. These results are discussed in terms of intracellular Ca²⁺-buffering and junctional sensitivity to Ca²⁺.