The Sertoli cell occluding junctions and gap junctions in mature and developing mammalian testis.
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Myoendothelial gap junctions: the gap is there, but does EDHF go through it?
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[Freeze-fractured and freeze-etched replicas of tight junctions and gap junctions in the citiary epithelium of rhesus monkeys (author's transl)].
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Animals↗
Regulation of gap junctional conductance.
Gap junctional conductance is regulated by the number of channels between coupled cells (the balance between formation and loss of these channels) and by the fraction of these channels that are open (gating mechanisms). A variety of treatments are known to affect junction formation. Adenosine 3',5'-cyclic monophosphate (cAMP) is involved in some cases, and protein synthesis may be required but precursor molecules can also exist. Junction removal occurs both by dispersion of particles and by internalization of junctional membrane. Factors promoting removal are not well understood. A variety of gating mechanisms exist. Coupling may be controlled by changes in conductance of nonjunctional membranes. Several kinds of voltage dependence of junctional conductance are known, but rat ventricular junctions at least are electrically linear. Cytoplasmic acidification decreases conductance of most gap junctions. Sensitivity in rat ventricular myocytes allows modulation of coupling by moderate changes near normal internal pH. Increasing intracellular Ca also decreases junctional conductance, but in the better studied cases sensitivity is much lower to Ca than H. A few data support low sensitivity to Ca in cardiac cells, but quantitative studies are lacking. Higher alcohols such as octanol block junctional conductance in a wide range of tissues including rat ventricular myocytes. An antibody to liver gap junctions blocks junctions between rat ventricular myocytes. Cross reactivity indicates at least partial homology between many gap junctions. Although differences among gap junctions are known, a general physiology is being developed, which may have considerable relevance to normal cardiac function and also to conduction disorders of that tissue.
Animals↗
The role of connexon aggregate fusion in gap junction growth.
Gap junctions are thought to grow in size by the incorporation of single connexons and by the fusion of connexon aggregates but the relative importance of these two growth mechanisms, particularly in the formation of large gap junctions, remains unclear. We have used a quantitative approach to distinguish between these two growth mechanisms and present evidence here that gap junctions in Drosophila melanogaster wing discs have a periodic, (approximately equal to 0.025 micron 2), multimodal areal distribution which suggests that gap junctions grow by aggregate fusion. Previously published gap junction areal frequency distributions from another invertebrate, Manduca sexta, and from several vertebrate animals also show periodic (approximately equal to 0.030 micron 2), multimodal areal distributions. We suggest that the interpeak spacings on the frequency distributions represent average areas of 'unit aggregates' of connexons and that the fusion of such unit aggregates with one another and with integral multiples of unit aggregates may be a general mechanism for gap junction growth. Unit aggregates in mammals and insects are similar in surface area rather than in connexon number suggesting that a common molecular mechanism, perhaps operating at the level of the plasma membrane, may be involved in their formation and stabilization.
Animals↗
Characteristic distribution of gap junctions in rat lacrimal gland in vivo and reconstruction of a gap junction in an in vitro model.
We investigated localization of the gap junction in rat lacrimal gland in vivo and in vitro using electron microscopy and immunostaining with anti-connexin32 (Cx32) monoclonal antibody (HAM8). In immunofluorescence study of lacrimal gland tissues, Cx32 protein appeared to exist not only at the intercellular borders of acinar cells, but also in the basal regions, where there apparently was no contact with adjacent acinar cells. Thin sectioning and immunoelectron microscopy revealed that lacrimal acinar cells formed autocellular gap junctions (reflexive gap junctions) in the basal regions and intercellular gap junctions with adjacent acinar cell membranes. In immunofluorescence study of primary culture, Cx32 protein was found on the free surfaces of isolated acinar cells at the early stage of culture. With culturing time, cell aggregates were formed. We observed Cx32 immunoreactivity between acinar cells in these aggregates, but not on their free surface. Electron microscopic study confirmed that these aggregates possessed intercellular gap junctions and morphologically differentiated acinar-like structures. However, reflexive gap junctions were not observed in these aggregates. In conclusion, lacrimal acinar cells form intercellular and reflexive gap junctions in vivo. On the other hand, the existence of an acinar-like structure and intercellular gap junctions indicates that acinar cells differentiated in vitro morphologically. The cells may communicate with each other through these junctions, organizing themselves into an acinar cell network in an in vitro situation.
Animals↗
Cell biology and protein composition of cardiac gap junctions.
The gap junctions that electrically couple mammalian myocardial cells have high (12,000-17,000/micron2)surface densities of channel-containing elements (connexons), undulating surfaces, and approximately hexagonally arrayed connexons disposed in small domains rotated with respect to one another. Optical diffraction combined with image processing of negatively stained isolated rabbit heart gap junctions shows six protein subunits surrounding the cell-to-cell channel and suggestive (but not conclusive) evidence for protein connections between connexons. Biochemical studies indicate that the six identical relative molecular weight (Mr) 47,000 subunits of mammalian cardiac gap junctions differ from liver gap junctions in the presence of a covalently bound Mr 17,500 cytoplasmic surface component that can be visualized electron microscopically in thin-sectioned and freeze-etched hearts. The cytoplasmic surface component is susceptible to cleavage by an alkaline serine protease released from mast cell granules by high ionic strength solutions (0.6 M KI) used to extract myofibrils during gap junction purification. Interlocking of connexons from coupled cells in the gap involves hydrogen bonding between protein subunits of the connexons.
Animals↗
Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of membrane pores: implications for the assembly of gap junctions.
Gap-junction channels provide a widespread intercellular signalling mechanism. They are constructed of a family of connexin membrane proteins that thread across the membrane four times and oligomerize to generate hexameric gap-junction hemichannels. Using an in vitro cell-free transcription/translation system, we demonstrate that connexin (Cx) 26, one of the smallest connexins, is integrated directly in a post-translational manner into plasma membranes. Protein-cleavage studies of Cx26 integrated into plasma membranes indicate a similar native transmembrane topography to that of Cx26 integrated co-translationally into microsomes. Cx26 integrated post-translationally into plasma membranes oligomerizes and, when incorporated into liposomes, provides permeability to ascorbic acid, suggesting that gap-junction hemichannels are generated. The results provide the basis of a novel alternative mechanism for spontaneous assembly in plasma membranes of Cx26 gap-junction hemichannels that occurs independently of the conventional biogenesis of gap junctions involving connexin trafficking and oligomerization via membrane components of the secretory pathway.
Animals↗
Rat endometrial stromal cells express the gap junction genes connexins 26 and 43 and form functional gap junctions during in vitro decidualization.
Gap junctions form between rat endometrial stromal cells as they undergo decidualization. We have examined the steady-state levels of the gap junction transcripts, connexins 26 and 43 (cx26 and cx43), during artificially induced decidualization in vivo and found that they have a temporal pattern similar to that observed in pregnancy. An in vitro model of decidualization was then used. Endometrial stromal cells from rat uteri sensitized for decidualization were cultured for 24, 48, or 72 h before total RNA was extracted and subjected to Northern blot analyses to determine the steady-state levels of cx26 and cx43 transcripts. The analyses revealed that cx26 transcript steady-state levels decreased, whereas those for cx43 increased, from 24 to 72 h. Using an anti-cx43 antibody, punctate immunofluorescent signals were observed around the periphery of the cells, suggesting that cx43 had been assembled into membrane plaques. The presence of functional gap junctions between the cells was determined in vitro by two dye-coupling methods: preloading and scrape-loading. Calcein (995 Da) and a membrane-bound dye, dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate (933 Da), were preloaded into 5% of the endometrial stromal cells before plating. The percentage of preloaded cells that transferred calcein to adjacent cells increased from 10% at 3 h after plating to 40% at 6 h. To determine whether or not cells maintain the ability to dye-couple throughout the culture period, carboxyfluorescein (CF; 376 Da) and rhodamine dextran (9.3 kDa) were introduced into cells by scraping the cells with a scalpel, and the distribution of dyes was determined 20 min later. In cells cultured for 24, 48, or 72 h, only CF was transferred to cells distal to the scrape line. The results from these experiments suggest that stromal cells can dye-couple throughout the culture period (3-72 h) and indicate that functional gap junctions form between endometrial stromal cells as they undergo decidualization in vitro.
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The effects of connexin phosphorylation on gap junctional communication.
Gap junctions are specialized membrane domains composed of collections of channels that directly connect neighboring cells providing for the cell-to-cell diffusion of small molecules, including ions, amino acids, nucleotides, and second messengers. Vertebrate gap junctions are composed of proteins encoded by the "connexin" gene family. In most cases examined, connexins are modified post-translationally by phosphorylation. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the connexin "lifecycle", such as the trafficking, assembly/disassembly, degradation, as well as, the gating of gap junction channels. Since connexin43 (Cx43) is widely expressed in tissues and cell lines, we understand the most about how it is regulated, and thus, connexin43 phosphorylation is a major focus of this review. Recent reports utilizing new methodologies combined with the latest genome information have shown that activation of several kinases including protein kinase A, protein kinase C, p34(cdc2)/cyclin B kinase, casein kinase 1, mitogen-activated protein (MAP) kinase and pp60(src) kinase can lead to phosphorylation at 12 of the 21 serine and two of the six tyrosine residues in the C-terminal region of connexin43. In several cases, use of site-directed mutants of these sites have shown that these specific phosphorylation events can be linked to changes in gap junctional communication.
Amino Acid Sequence↗
Innexins: members of an evolutionarily conserved family of gap-junction proteins.
Gap junctions are clusters of intercellular channels that provide cells, in all metazoan organisms, with a means of communicating directly with their neighbours. Surprisingly, two gene families have evolved to fulfil this fundamental, and highly conserved, function. In vertebrates, gap junctions are assembled from a large family of connexin proteins. Innexins were originally characterized as the structural components of gap junctions in Drosophila, an arthropod, and the nematode Caenorhabditis elegans. Since then, innexin homologues have been identified in representatives of the other major invertebrate phyla and in insect-associated viruses. Intriguingly, functional innexin homologues have also been found in vertebrate genomes. These studies have informed our understanding of the molecular evolution of gap junctions and have greatly expanded the numbers of model systems available for functional studies. Genetic manipulation of innexin function in relatively simple cellular systems should speed progress not only in defining the importance of gap junctions in a variety of biological processes but also in elucidating the mechanisms by which they act.
Amino Acid Sequence↗
The use of antibodies to gap junction protein to explore the role of gap junctional communication during development.
Antibodies raised against the major 27 kDa protein electrophoretically eluted from isolated gap junctions and affinity purified against the antigen have been used to explore the role of communication through gap junctions in the early amphibian and mouse embryos. In both species, injection of the antibodies into one cell completely blocks both dye transfer and electrical coupling between cells connected by gap junctions. In the amphibian embryo the generation of a communication-incompetent clone of cells leads to patterning defects in the region derived from the antibody-injected cell. In the mouse embryo, blocking cell-to-cell communication leads to decompaction of the communication-incompetent cells. The possible significance of these findings in relation to development in general and to the organization of the first transporting epithelia to appear during development is discussed.
Animals↗
Short- and long-term regulation of rat carotid body gap junctions by cAMP. Identification of connexin43, a gap junction subunit.
Intact and cultured carotid bodies (CBs) of the rat were used in this study. Applications of membrane-permeant db-cAMP to cultured carotid bodies increased electric coupling between most glomus cells (increasing junctional conductance) probably by opening preformed intercellular channels. This a short-term effect of the nucleotide, increasing gating between glomus cells. When cultures and intact carotid bodies were treated with membrane-permeant 8Br-cAMP for 3 h or more (to increase cytosolic cAMP), there was enhanced gap junction formation and better dye spread between carotid body cells. Connexin43 (CX43) was identified by immunocytochemical methods as forming part of the intercellular channels between carotid body cells, and the expression of Cx43 increased by cAMP. This is a long-term effect, inducing the formation of gap junctions. Thus, cAMP had short and long-term effects on the intercellular junctions of the carotid body. Long-term formation of gap junctions may be important in modulating carotid body functions during stimulation by chronic hypoxia.
8-Bromo Cyclic Adenosine Monophosphate↗
Cytoplasmic surface and intramembrane components of rat heart gap junctional proteins.
Gap junctions were purified from rat hearts in the presence of absence of proteolysis inhibitors and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy of thin sections. In absence of proteolysis inhibitors or in presence of ethylenediaminetetraacetic acid or leupeptin, gap junctions contained a single major protein band at relative molecular weight (Mr) 29,500 and minor bands at Mr 44,000-47,000, 17,750, and 16,500 and showed smooth cytoplasmic surfaces in electron micrographs. SDS-PAGE of junctions prepared with phenylmethylsulfonylfluoride (PMSF) showed markedly decreased intensity of the Mr 29,500 band and increased intensity of bands at Mr 44,000, 45,500, and 47,000; electron microscopy of these gap junctions showed presence of a fuzzy layer on their cytoplasmic surfaces. Urea (8 M) could not remove this fuzzy layer. In electron micrographs of rat ventricular myocytes, cytoplasmic surfaces of gap junctions were fuzzy. We conclude that rat heart gap junction protein consists of an intramembrane component (Mr 29,500) that extends into the "gap" and a cytoplasmic surface component (Mr 14,500-17,500) that corresponds to the fuzzy layer and is hydrolyzable by a serine protease.
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Functional assembly of gap junction conductance in lipid bilayers: demonstration that the major 27 kd protein forms the junctional channel.
Gap junctions isolated from rat liver were incorporated into planar lipid bilayers. A channel activity that was directly dependent on voltage was recorded. Changes of pH and (Ca2+) had no direct effect on channel activity; however, they modulated the voltage-dependent gating of the gap junction channels differently. Single-channel fluctuations showed large scatter with peak amplitudes of 140 and 280 picoSiemmens in 0.1 M NaCl. The major protein of gap junctions (Mr of 27 kd) was also reconstituted into bilayers, giving channel properties similar to those of intact gap junctions. Polyclonal antibodies specific for this protein caused inhibition of the junctional conductance in bilayers. These data provide direct evidence that the 27 kd protein is the molecular species responsible for gap junction communication between cells.
Animals↗
Expression of gap-junctional protein (connexin 43 or alpha 1 gap junction) is down-regulated at the transcriptional level during adipocyte differentiation of H-1/A marrow stromal cells.
Bone marrow stromal cells are requisite for the proliferation of hematopoietic cells and communicate with each other via gap junctions. Marrow stromal cells expressed connexin 43, but not connexin 32. H-1/A, a murine marrow stromal cell line, underwent adipocyte differentiation at confluence, and expressed the 3.0 kilobase mRNA species of connexin 43 before differentiation. H-1/A cells studied with the dye-transfer method showed gap-junctional communication with adjacent cells but lost this communication during differentiation. The connexin 43 transcripts in H-1/A cells were down-regulated before the expression of glycero-monophosphate dehydrogenase was induced. Loss of gap-junctional communication was regulated at the mRNA level of connexin 43. Connexin 43 expression was down-regulated at the transcriptional level.
Adipose Tissue↗
Gap junctions in several tissues share antigenic determinants with liver gap junctions.
Using affinity-purified antibodies against mouse liver gap junction protein (26 K), discrete fluorescent spots were seen by indirect immunofluorescence labelling on apposed membranes of contiguous cells in several mouse and rat tissues: pancreas (exocrine part), kidney, small intestine (epithelium and circular smooth muscle), Fallopian tube, endometrium, and myometrium of delivering rats. No reaction was seen on sections of myocardium, ovaries and lens. Specific labelling of gap junction plaques was demonstrated by immunoelectron microscopy on ultrathin frozen sections through liver and the exocrine part of pancreas after treatment with gold protein A. Weak immunoreactivity was found on the endocrine part of the pancreas (i.e., Langerhans islets) after glibenclamide treatment of mice and rats, which causes an increase of insulin secretion and of the size as well as the number of gap junction plaques in cells of Langerhans islets. Furthermore, the affinity purified anti-liver 26 K antibodies were shown by immunoblot to react with proteins of similar mol. wt. in pancreas and kidney membranes. Taken together these results suggest that gap junctions from several, morphogenetically different tissues have specific antigenic sites in common. The different extent of specific immunoreactivity of anti-liver 26 K antibodies with different tissues is likely due to differences in size and number of gap junctions although structural differences cannot be excluded.
Animals↗
Functional role of the carboxyl terminal domain of human connexin 50 in gap junctional channels.
Gap junction channels formed by connexin 50 (Cx50) are critical for maintenance of lens transparency. Because the C-terminus of Cx50 can be cleaved post-translationally, we hypothesized that channels formed by the truncated Cx50 exhibit altered properties or regulation. We used the dual whole-cell patch-clamp technique to investigate the macroscopic and single-channel properties of gap junctional channels formed by wild-type human Cx50 and a truncation mutant (Cx50A294stop) after transfection of N2A cells. Our results show that wild-type Cx50 formed functional gap junctional channels. The macroscopic Gjss-Vj relationship was well described by a Boltzmann equation with A of 0.10, V0 of 43.8 mV and Gjmin of 0.23. The single-channel conductance was 212 +/- 5 pS. Multiple long-lasting substates were observed with conductances ranging between 31 and 80 pS. Wild-type Cx50 gap junctional channels were reversibly blocked when pHi was reduced to 6.3. Truncating the C-terminus at amino acid 294 caused a loss of pHi sensitivity, but there were no significant changes in single-channel current amplitude or Gjss-Vj relationship. These results suggest that the C-terminus of human Cx50 is involved in pHi sensitivity, but has little influence over single-channel conductance, voltage dependence, or gating kinetics.
Amino Acid Sequence↗