Regulation of gap junction intercellular communication by the ubiquitin system

Intercellular communication via gap junctions plays a critical role in numerous cellular processes, including the control of cell growth and differentiation, maintenance of tissue homeostasis and embryonic development. Gap junctions are aggregates of intercellular channels that enable adjacent cells in solid tissues to directly exchange ions and small molecules. These channels are formed by a family of integral membrane proteins called connexins, of which the best studied is connexin43. Connexins have a high turnover rate in most tissue types, and degradation of connexins is considered to be a tightly regulated process. Post-translational modification of connexins by ubiquitin is emerging as an important event in the regulation of connexin degradation. Ubiquitination is involved in endoplasmic reticulum-associated degradation of connexins as well as in trafficking of connexins to lysosomes. At both the endoplasmic reticulum and the plasma membrane, ubiquitination of connexins is strongly affected by changes in the extracellular environment. There is increasing evidence that the regulation of connexin ubiquitination might be an important mechanism for rapidly modifying the level of functional gap junctions at the plasma membrane, under both normal and pathological conditions. This review discusses the current knowledge about the regulation of intercellular communication via gap junctions by ubiquitination of connexins.     

Multiple connexin proteins in single intercellular channels: Connexin compatibility and functional consequences

In vertebrates, the protein subunits of intercellular channels found in gap junctions are encoded by a family of genes called connexins. These channels span two plasma membranes and result from the association of two half channels, or connexons, which are hexameric assemblies of connexins. Physiological analysis of channel formation and gating has revealed unique patterns of connexin-connexin interaction, and uncovered novel functional characteristics of channels containing more than one type of connexin protein. Structure-function studies have further demonstrated that unique domains within connexins participate in the regulation of different functional properties of intercellular channels. Thus, gap junctional channels can contain more than one connexin, and this structural heterogeneity has functional consequencesin vitro. Moreover, emerging evidence for the existence of intercellular channels containing multiple connexins in native tissues suggests that the functional diversity generated by connexin-connexin interaction could contribute to complex communication patterns that have been observedin vivo.     

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.     

Degradation of Connexins Through the Proteasomal, Endolysosomal and Phagolysosomal Pathways

Connexins comprise gap junction channels, which create a direct conduit between the cytoplasms of adjacent cells and provide for intercellular communication. Therefore, the level of total cellular connexin protein can have a direct influence on the level of intercellular communication. Control of connexin protein levels can occur through different mechanisms during the connexin life cycle, such as by regulation of connexin gene expression and turnover of existing protein. The degradation of connexins has been extensively studied, revealing proteasomal, endolysosomal and more recently autophagosomal degradation mechanisms that modulate connexin turnover and, subsequently, affect intercellular communication. Here, we review the current knowledge of connexin degradation pathways.     

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.     

Pathways and control of connexin oligomerization

Connexins form gap junction channels that link neighboring cells into an intercellular communication network. Many cells that express multiple connexins produce heteromeric channels containing at least two connexins, which provides a means to fine tune gap junctional communication. Formation of channels by multiple connexins is controlled at two levels: by inherent structural compatibilities that enable connexins to hetero-oligomerize and by cellular mechanisms that restrict the formation of heteromers by otherwise compatible connexins. Here, I discuss roles for secretory compartments beyond the endoplasmic reticulum in connexin oligomerization and evidence that suggests that membrane microdomains help regulate connexin trafficking and assembly.     

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 approximately 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.     

Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins

Gap junctions are collections of intercellular channels composed of structural proteins called connexins (Cx). We have examined the functional interactions of the three rodent connexins present in the lens, Cx43, Cx46, and Cx50, by expressing them in paired Xenopus oocytes. Homotypic channels containing Cx43, Cx46, or Cx50 all developed high conductance. heterotypic channels composed of Cx46 paired with either Cx43 or Cx50 were also well coupled, whereas Cx50 did not form functional channels with Cx43. We also examined the functional response of homotypic and heterotypic channels to transjunctional voltage and cytoplasmic acidification. We show that all lens connexins exhibited sensitivity to cytoplasmic acidification as well as to voltage, and that voltage-dependent closure of heterotypic channels for a given connexin was dramatically influenced by its partner connexins in the adjacent cell. Based on the observation that Cx43 can discriminate between Cx46 and Cx50, we investigated the molecular determinants that specify compatibility by constructing chimeric connexins from portions of Cx46 and Cx50 and testing them for their ability to form channels with Cx43. When the second extracellular (E2) domain in Cx46 was replaced with the E2 of Cx50, the resulting chimera could no longer form heterotypic channels with Cx43. A reciprocal chimera, where the E2 of Cx46 was inserted into Cx50, acquired the ability to functionally interact with Cx43. Together, these results demonstrate that formation of intercellular channels is a selective process dependent on the identity of the connexins expressed in adjacent cells, and that the second extracellular domain is a determinant of heterotypic compatibility between connexins.     

Cloning and functional expression of a novel human connexin-25 gene

Gap junctions are intercellular, water-filled channels composed of transmembrane proteins called connexins, six of which are arranged radially and dock with six homologous proteins in an adjacent cell to form an approximate 16 A pore. Through this pore cell-to-cell transfer of small water-soluble molecules up to about 1000 daltons occurs along concentration gradients. Connexins comprise a multigene family that share consensus sequences in the trans-membrane domains and the first and second extracellular loops. Comparison of the protein sequences of known human connexins with the draft nucleotide sequence of the human genome revealed two clones from chromosome 6 which showed strong similarity to highly conserved connexin sequences. Detailed analysis revealed the presence of a 672 nt open reading frame in these clones, encoding a 223 amino acid polypeptide with a predicted molecular weight of about 25 kD. This is smaller than other known human connexins. The ORF of the potential connexin25 was amplified by semi-nested PCR using human genomic DNA as a template. To confirm that this new gene encodes a connexin, Cx25 was transfected into a gap junction deficient subclone of the human HeLa cell line. After selection of transformants, cells were microinjected with the fluorescent dye Lucifer yellow. Transfectants but not controls successfully transferred dye, demonstrating that this new gene encodes a functional connexin.     

Editorial,Introduction and Acknowledgements

Gap junctions arc intercellular, water-filled channels composed of transmembrane proteins called connexins, six of which are arranged radially and dock with six homologous proteins in an adjacent cell to form an approximate 16 A pore. Through this pore cell-to-cell transfer of small water-soluble molecules up to about 1000 daltons occurs along concentration gradients. Connexins comprise a multigene family that share consensus sequences in the trans-membrane domains and the first and second extracellular loops. Comparison of the protein sequences of known human connexins with the draft nucleotide sequence of the human genome revealed two clones from chromosome 6 which showed strong similarity to highly conserved connexin sequences. Detailed analysis revealed the presence of a 672 nt open reading frame in these clones, encoding a 223 amino acid polypeptide with a predicted molecular weight of about 25 kD. This is smaller than other known human connexins. The ORF of the potential connexin25 was amplified by semi-nested PCR using human genomic DNA as a template. To confirm that this new gene encodes a connexin, Cx25 was transfected into a gap junction deficient subclone of the human HeLa cell line. After selection of transformants, cells were microinjected with the fluorescent dye Lucifer yellow. Transfectants but not controls successfully transferred dye, demonstrating that this new gene encodes a functional connexin.     

Cloning and Functional Expression of a Novel Human Connexin-25 Gene

Gap junctions arc intercellular, water-filled channels composed of transmembrane proteins called connexins, six of which are arranged radially and dock with six homologous proteins in an adjacent cell to form an approximate 16 A pore. Through this pore cell-to-cell transfer of small water-soluble molecules up to about 1000 daltons occurs along concentration gradients. Connexins comprise a multigene family that share consensus sequences in the trans-membrane domains and the first and second extracellular loops. Comparison of the protein sequences of known human connexins with the draft nucleotide sequence of the human genome revealed two clones from chromosome 6 which showed strong similarity to highly conserved connexin sequences. Detailed analysis revealed the presence of a 672 nt open reading frame in these clones, encoding a 223 amino acid polypeptide with a predicted molecular weight of about 25 kD. This is smaller than other known human connexins. The ORF of the potential connexin25 was amplified by semi-nested PCR using human genomic DNA as a template. To confirm that this new gene encodes a connexin, Cx25 was transfected into a gap junction deficient subclone of the human HeLa cell line. After selection of transformants, cells were microinjected with the fluorescent dye Lucifer yellow. Transfectants but not controls successfully transferred dye, demonstrating that this new gene encodes a functional connexin.     

Functional redundancy and compensation among members of gap junction protein families?

Gap junctions are intercellular conduits for small molecules made up by protein subunits called connexins. A large number of connexin genes were found in mouse and man, and most cell types express several connexins, lending support to the view that redundancy and compensation among family members exist. This review gives an overview of the current knowledge on redundancy and functional compensation - or lack thereof. It takes into account the different properties of connexin subunits which comprise gap junctional intercellular channels, but also the compatibility of connexins in gap junctions. Most insight has been gained by the investigation of mice deficient for one or more connexins and transgenic mice with functional replacement of one connexin gene by another. Most single deficient mice show phenotypical alterations limited to critical developmental time points or to specific organs and tissues, while mice doubly deficient for connexins expressed in the same cell type usually show more severe phenotypical alterations. Replacement of a connexin by another connexin in some cases gave rise to rescue of phenotypical alterations of connexin deficiencies, which were restricted to specific tissues. In many tissues, connexin substitution did not restore phenotypical alterations of connexin deficiencies, indicating that connexins are specialized in function. In some cases, fatal consequences arose from the replacement. The current consensus gained from such studies is that redundancy and compensation among connexins exists at least to a limited extent. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle

Summary Gap junctions contain intercellular channels which are formed by members of a group of related proteins called connexins. Connexins contain conserved transmembrane and extracellular domains, but unique cytoplasmic regions which may provide connexin-specific physiologic properties. We used polymerase chain reaction (PCR) amplification and cDNA library screening to clone DNA encoding a novel member of this gene family, rat connexin40 (Cx40). The derived rat Cx40 polypeptide contains 356 amino acids, with a predicted molecular mass of 40,233 Da. Sequence comparisons suggest that Cx40 is the mammalian homologue of chick connexin42, but it has predicted cytoplasmic regions that differ from previously described mammalian connexins. Southern blots of rat genomic DNA suggest that Cx40 is encoded by a single copy gene containing no introns within its coding region. Northern blots demonstrate that Cx40 is expressed in multiple tissues (including lung, heart, uterus, ovary, and blood vessels) and in primary cultures and established lines of vascular smooth muscle cells. Cx40 is coexpressed with connexin43 in several cell types, including A7r5 cells, which contain two physiologically distinct gap junctional channels. To demonstrate that Cx40 could form functional channels, we stably transfected communication-deficient Neuro2A cells with Cx40 DNA. These Cx40-transfected cells showed intercellular passage of microinjected Lucifer yellow CH. The expression of multiple connexins (such as Cx40 and Cx43) by a single cell may provide a mechanism by which cells regulate intercellular coupling through the formation of multiple channels     

Connexin and pannexin as modulators of myocardial injury

Multicellular organisms have developed a variety of mechanisms that allow communication between their cells. Whereas some of these systems, as neurotransmission or hormones, make possible communication between remote areas, direct cell-to-cell communication through specific membrane channels keep in contact neighboring cells. Direct communication between the cytoplasm of adjacent cells is achieved in vertebrates by membrane channels formed by connexins. However, in addition to allowing exchange of ions and small metabolites between the cytoplasms of adjacent cells, connexin channels also communicate the cytosol with the extracellular space, thus enabling a completely different communication system, involving activation of extracellular receptors. Recently, the demonstration of connexin at the inner mitochondrial membrane of cardiomyocytes, probably forming hemichannels, has enlarged the list of actions of connexins. Some of these mechanisms are also shared by a different family of proteins, termed pannexins. Importantly, these systems allow not only communication between healthy cells, but also play an important role during different types of injury. The aim of this review is to discuss the role played by both connexin hemichannels and pannexin channels in cell communication and injury. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Roles and regulation of lens epithelial cell connexins

The avascular lens of the eye is covered anteriorly by an epithelium containing nucleated, metabolically active cells. This epithelium contains the first lens cells to encounter noxious external stimuli and cells that can develop compensatory or protective responses. Lens epithelial cells express the gap junction proteins, connexin43 (Cx43) and connexin50 (Cx50). Cx43 and Cx50 form gap junction channels and hemichannels with different properties. Although they may form heteromeric hemichannels, Cx43 and Cx50 probably do not form heterotypic channels in the lens. Cx50 channels make their greatest contribution to intercellular communication during the early postnatal period; subsequently, Cx43 becomes the predominant connexin supporting intercellular communication. Although epithelial Cx43 appears dispensable for lens development, Cx50 is critical for epithelial cell proliferation and differentiation. Cx43 and Cx50 hemichannels and gap junction channels are regulated by multiple different agents. Lens epithelial cell connexins contribute to both normal lens physiology and pathology.     

Adenoviral delivery of human connexin37 induces endothelial cell death through apoptosis

Gap junction channels formed of connexins directly link the cytoplasm of adjacent cells and have been implicated in intercellular signaling that may regulate the functions of vascular cells. To facilitate connexin manipulation and analysis of their roles in adult endothelial cells, we developed adenoviruses containing the vascular connexins (Cx37, Cx40, and Cx43). We infected cultured human umbilical vein endothelial cells with control or connexin adenoviruses. Connexin expression was verified by immunoblotting and immunofluorescence. Infection with the Cx37 adenovirus (but not control or other connexin adenoviruses) led to a dose-dependent death of the endothelial cells that was partially antagonized by the gap junction blocker alpha-glycyrrhetinic acid and altered the intercellular transfer of Lucifer yellow and neurobiotin. Cell morphology, Annexin V and TUNEL staining, and caspase 3 assays all implicated apoptosis in the cell death. These data suggest that connexin-specific alterations of intercellular communication may modulate endothelial cell growth and death.     

Connexin Genes in the Mouse and Human Genome

Gap junctions serve for direct intercellular communication by docking of two hemichannels in adjacent cells thereby forming conduits between the cytoplasmic compartments of adjacent cells. Connexin genes code for subunit proteins of gap junction channels and are members of large gene families in mammals. So far, 17 connexin (Cx) genes have been described and characterized in the murine genome. For most of them, orthologues in the human genome have been found (see White and Paul 1999; Manthey et al. 1999; Teubner et al. 2001; Söhl et al. 2001). We have recently performed searches for connexin genes in murine and human gene libraries available at EMBL/Heidelberg, NCBI and the Celera company that have increased the number of identified connexins to 19 in mouse and 20 in humans. For one mouse connexin gene and two human connexin genes we did not find orthologues in the other genome. Here we present a short overview on distinct connexin genes which we found in the mouse and human genome and which may include all members of this gene family, if no further connexin gene will be discovered in the remaining non-sequenced parts (about 1-5%) of the genomes.     

Connexin genes in the mouse and human genome

Gap junctions serve for direct intercellular communication by docking of two hemichannels in adjacent cells thereby forming conduits between the cytoplasmic compartments of adjacent cells. Connexin genes code for subunit proteins of gap junction channels and are members of large gene families in mammals. So far, 17 connexin (Cx) genes have been described and characterized in the murine genome. For most of them, orthologues in the human genome have been found (see White and Paul 1999; Manthey et al. 1999; Teubner et al. 2001; S?hl et al. 2001). We have recently performed searches for connexin genes in murine and human gene libraries available at EMBL/Heidelberg, NCBI and the Celera company that have increased the number of identified connexins to 19 in mouse and 20 in humans. For one mouse connexin gene and two human connexin genes we did not find orthologues in the other genome. Here we present a short overview on distinct connexin genes which we found in the mouse and human genome and which may include all members of this gene family, if no further connexin gene will be discovered in the remaining non-sequenced parts (about 1-5%) of the genomes.     

Gap junction blockage interferes with neuronal and astroglial differentiation of mouse P19 embryonal carcinoma cells

During embryonic development, cells not only increase in number, they also undergo specialization and differentiate into diverse cell types that are organized into different tissues and organs. Nervous system development, for example, involves a complex series of events such as neuronal and astroglial differentiation that are coordinated among adjacent cells. The organization of growth and differentiation may be mediated, at least partly, by exchange of small ions and molecules via intercellular gap junction channels. These structures are mode of connexons (hemichannels), which are hexameric assemblies of the gap junction proteins, connexins. We investigated the role of intercellular communication in neuronal and astroglial differentiation by using a gap junction blocking agent, carbenoxolone (CBX), in comparison to its inactive (control) analog, glycyrrhizic acid (GZA). We used the mouse P19 embryonal carcinoma cell line, which differentiates into neurons and astrocytes upon retinoic acid (RA) induction. Our results show that both GZA- and CBX-treated cells express alpha 1 connexin (connexin43). The level of alpha 1 connexin decreases upon RA induction. CBX treated cells show significant reduction in both neuronal (5-fold) and astrocytic (13-fold) differentiation compared with those of control. These results clearly indicate that the blockage of gap junction-mediated intercellular communication interferes with differentiation of P19 cells into neurons and astrocytes.