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.     

An Update on Connexin Genes and their Nomenclature in Mouse and Man

Gap junctions, composed of connexin protein subunits, allow direct communication through conduits between neighboring cells. Twenty and twenty-one members of the connexin gene family are likely to be expressed in the mouse and human genome, respectively, 19 of which can be grouped into sequence-orthologous pairs. Their gene structure appears to be relatively simple. In most cases, an untranslated exon1 is separated by an intron of different lengh from exon2 that includes the uninterrupted coding region and the 3'-untranslated region. However, there are several exceptions to this scheme, since some mouse connexin genes contain different 5'-untranslated regions spliced either in an alternative and/or consecutive manner. Additionally, in at least 3 mouse and human connexin genes (mCx36, mCx39, mCx57 and hCx31.3, hCx36, as well as hCx40.1) the reading frame is spliced together from 2 different exons. So far, there are two nomenclatures to classify the known connexin genes: The “Gja/Gjb” nomenclature, as it is currently adopted by the NCBI data base, contains some inconsistencies compared to the “Cx” nomenclature. Here we suggest some minor corrections to co-ordinate the “Gja/Gjb” nomenclature with the “Cx” nomenclature. Furthermore, this short review contains an update on phenotypic correlations between connexin deficient mice and patients bearing mutations in their orthologous connexin genes.     

Structural and functional diversity of connexin genes in the mouse and human genome

Gap junctions are clustered channels between contacting cells through which direct intercellular communication via diffusion of ions and metabolites can occur. Two hemichannels, each built up of six connexin protein subunits in the plasma membrane of adjacent cells, can dock to each other to form conduits between cells. We have recently screened mouse and human genomic data bases and have found 19 connexin (Cx) genes in the mouse genome and 20 connexin genes in the human genome. One mouse connexin gene and two human connexin genes do not appear to have orthologs in the other genome. With three exceptions, the characterized connexin genes comprise two exons whereby the complete reading frame is located on the second exon. Targeted ablation of eleven mouse connexin genes revealed basic insights into the functional diversity of the connexin gene family. In addition, the phenotypes of human genetic disorders caused by mutated connexin genes further complement our understanding of connexin functions in the human organism. In this review we compare currently identified connexin genes in both the mouse and human genome and discuss the functions of gap junctions deduced from targeted mouse mutants and human genetic disorders.     

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.     

Injury of skeletal muscle and specific cytokines induce the expression of gap junction channels in mouse dendritic cells

Dendritic cells (DCs) in culture express at least connexin43, a protein subunit of gap junctions, and form gap junction channels, which could be important for T-cells activation. Here, we evaluated whether DCs express connexins in vivo and also to identify components of their microenvironment that regulate the functional expression of gap junctions. In vivo studies were performed in lymph nodes of mice under control conditions or after skeletal muscle damage. In double immunolabeling studies, connexin45 was frequently detected in DEC205(+) DCs in lymph nodes of control animals, whereas connexin43 was rarely found in DCs. However, connexin43 was upregulated in DCs after skeletal muscle damage. Upregulation of connexin43 gene expression by tissue damage was also confirmed in mice carrying a beta-galactosidase reporter gene in a connexin43 allele. The effect of several cytokines on the expression of functional gap junctions between cultured DCs was also tested. Under control conditions, cultured DCs did not communicate via gap junctions. However, after treatment with keratinocyte-conditioned medium or cytokine mixtures containing at least TNF-alpha and IL-1beta, they became transiently coupled through a pathway sensitive to octanol, a gap junction blocker. Cellular coupling induced by effective cytokine mixtures was prevented by IL-6. Single cytokines (TNF-alpha, IL-1beta, IFN-gamma, or IL-6) or other mixtures than the described above did not induce coupling via gap junctions. Increased levels of connexin43 and connexin45 protein and mRNA accompanied the appearance of cellular coupling. These studies provide demonstration of connexin expression and regulation by specific danger signals in DCs.     

An Update on Connexin Genes and their Nomenclature in Mouse and Man

Gap junctions, composed of connexin protein subunits, allow direct communication through conduits between neighboring cells. Twenty and twenty-one members of the connexin gene family are likely to be expressed in the mouse and human genome, respectively, 19 of which can be grouped into sequence-orthologous pairs. Their gene structure appears to be relatively simple. In most cases, an untranslated exon1 is separated by an intron of different lengh from exon2 that includes the uninterrupted coding region and the 3′-untranslated region. However, there are several exceptions to this scheme, since some mouse connexin genes contain different 5′-untranslated regions spliced either in an alternative and/or consecutive manner. Additionally, in at least 3 mouse and human connexin genes (mCx36, mCx39, mCx57 and hCx31.3, hCx36, as well as hCx40.1) the reading frame is spliced together from 2 different exons. So far, there are two nomenclatures to classify the known connexin genes: The “Gja/Gjb” nomenclature, as it is currently adopted by the NCBI data base, contains some inconsistencies compared to the “Cx” nomenclature. Here we suggest some minor corrections to co-ordinate the “Gja/Gjb” nomenclature with the “Cx” nomenclature. Furthermore, this short review contains an update on phenotypic correlations between connexin deficient mice and patients bearing mutations in their orthologous connexin genes.     

An update on connexin genes and their nomenclature in mouse and man

Gap junctions, composed of connexin protein subunits, allow direct communication through conduits between neighboring cells. Twenty and twenty-one members of the connexin gene family are likely to be expressed in the mouse and human genome, respectively, 19 of which can be grouped into sequence-orthologous pairs. Their gene structure appears to be relatively simple. In most cases, an untranslated exon1 is separated by an intron of different lengh from exon2 that includes the uninterrupted coding region and the 3'-untranslated region. However, there are several exceptions to this scheme, since some mouse connexin genes contain different 5'-untranslated regions spliced either in an alternative and/or consecutive manner. Additionally, in at least 3 mouse and human connexin genes (mCx36, mCx39, mCx57 and hCx31.3, hCx36, as well as hCx40.1) the reading frame is spliced together from 2 different exons. So far, there are two nomenclatures to classify the known connexin genes: The "Gja/Gjb" nomenclature, as it is currently adopted by the NCBI data base, contains some inconsistencies compared to the "Cx" nomenclature. Here we suggest some minor corrections to co-ordinate the "Gja/Gjb" nomenclature with the "Cx" nomenclature. Furthermore, this short review contains an update on phenotypic correlations between connexin deficient mice and patients bearing mutations in their orthologous connexin genes.     

Characterization of connexin30.3-deficient mice suggests a possible role of connexin30.3 in olfaction

We have generated connexin30.3-deficient mice in which the coding region of the connexin30.3 gene was replaced by the lacZ reporter gene. The expression pattern of this connexin was characterized using beta-galactosidase staining and immunoblot analyses. In skin, beta-galactosidase/connexin30.3 protein was expressed in the spinous and granulous layers of the epidermis. Specific beta-galactosidase/connexin30.3 expression was also detected in the thin ascending limb of Henle's loop in the kidney. In addition, we found beta-galactosidase/connexin30.3 in progenitor cells of the olfactory epithelium and in a subpopulation of cells in the apical layer of the vomeronasal organ. Connexin30.3-deficient mice were fertile and displayed no abnormalities in the skin or in the chemosensory systems. Furthermore, they showed normal auditory thresholds as measured by brain stem evoked potentials. These mice did, however, exhibit reduced behavioural responses to a vanilla scent.     

Contribution of VH Replacement Products in Mouse Antibody Repertoire

V_H replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3′ end of a rearranged V_H gene and the 23-bp RSS from an upstream unrearranged V_H gene. Due to the location of the cRSS, V_H replacement leaves a short stretch of nucleotides from the previously rearranged V_H gene at the newly formed V-D junction, which can be used as a marker to identify V_H replacement products. To determine the contribution of V_H replacement products to mouse antibody repertoire, we developed a Java-based V_H Replacement Footprint Analyzer (V_HRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify V_H replacement products. The overall frequency of V_H replacement products in these IgH genes is 5.29% based on the identification of pentameric V_H replacement footprints at their V-D junctions. The identified V_H replacement products are distributed similarly in IgH genes using most families of V_H genes, although different families of V_H genes are used differentially. The frequencies of V_H replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified V_H replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of V_H replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.     

Role of connexin (gap junction) genes in cell growth control and carcinogenesis

Gap junctional intercellular communication (GJIC) is considered to play a key role in the maintenance of tissue independence and homeostasis in multicellular organisms by controlling the growth of GJIC-connected cells. Gap junction channels are composed of connexin molecules and, so far, more than a dozen different connexin genes have been shown to be expressed in mammals. Reflecting the importance of GJIC in various physiological functions, deletion of different connexin genes from mice results in various disorders, including cancers, heart malformation or conduction abnormality, cataract, etc. The possible involvement of aberrant GJIC in abnormal cell growth and carcinogenesis has long been postulated and recent studies in our own and other laboratories have confirmed that expression and function of connexin genes play an important role in cell growth control. Thus, almost all malignant cells show altered homologous and/or heterologous GJIC and are often associated with aberrant expression or localization of connexins. Aberrant localization of connexins in some tumour cells is associated with lack of function of cell adhesion molecules, suggesting the importance of cell-cell recognition for GJIC. Transfection of connexin genes into tumorigenic cells restores normal cell growth, supporting the idea that connexins form a family of tumour-suppressor genes. Some studies also show that specific connexins may be necessary to control growth of specific cell types. We have produced various dominant-negative mutants of Cx26, Cx32 and Cx43 and showed that some of them prevent the growth control exerted by the corresponding wild-type genes. However, we have found that connexins 32, 37 and 43 genes are rarely mutated in tumours. In some of these studies, we noted that connexin expression per se, rather than GJIC level, is more closely related to growth control, suggesting that connexins may have a GJIC-independent function. We have recently created a transgenic mouse strain in which a mutant Cx32 is specifically overexpressed in the liver. Studies with such mice indicate that Cx32 plays a key role in liver regeneration after partial hepatectomy. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through GJIC. Recently, we and others have shown that GJIC is responsible for the bystander effect seen in HSV-tk/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.     

Conditional allelic replacement applied to genes encoding the histone variant H3.3 in the mouse

Post‐translational modifications to residues in core histones convey epigenetic information. Their function can be evaluated in amino acid substitution mutants, although to date this method has not been used in mice. To this end, we have evaluated gene targeting vectors designed for Cre recombinase‐mediated conditional allelic replacement at the two unlinked genes encoding the histone variant H3.3. The conditional alleles consist of an uninterrupted wild‐type H3.3 coding sequence upstream of a desired alternative or proxy coding sequence. The arrangement of two loxP sites allows Cre‐mediated replacement of the wild‐type coding sequence with the proxy. To demonstrate proof of principle, at each locus we replaced the wild‐type coding sequence with a fluorescent reporter. This produced null alleles that will be useful to analyse the effects of H3.3 deficiency in development. Each targeting vector can readily be retrofitted with a proxy coding sequence encoding a modified H3.3 protein. Such vectors will allow for the conditional substitution of specific residues in order to dissect the roles of H3.3 post‐translational modifications in development and disease. genesis, 51:142?146, 2013. © 2013 Wiley Periodicals, Inc.     

The color of mice: in the light of GFP-variant reporters

The mouse currently represents the premier model organism for mammalian genetic studies. Over the past decade the production of targeted and transgenic lines of mice has become commonplace, with current technology allowing the creation of mutations at base pair resolution. Such genome modifications are becoming increasingly elaborate and often incorporate gene-based reporters for tagging different cellular populations. Until recently, lacZ, the bacterial beta-galactosidase gene has been the marker of choice for most studies in the mouse. However, over the past 3 years another valuable reporter has emerged, and its attractiveness is reflected by an explosion in its use in mice. Green fluorescent protein (GFP), a novel autofluorescent genetic reporter derived from the bioluminescent jellyfish Aequorea victoria, currently represents a unique alternative to other gene-based reporters in that its visualization is non-invasive and so can be monitored in real-time in vitro or in vivo. It has the added advantage that it can be quantified by, for example, flow cytometry, confocal microscopy, and fluorometric assays. Several mutants of the original wild-type GFP gene that improve thermostability and fluorescence have been engineered. Enhanced GFP is one such variant, which has gained popularity for use in transgenic or targeted mice. Moreover, various GFP spectral variants have also been developed, and two of these novel color variants, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), can also be used in mice. Since the spectral profiles of the ECFP and EYFP color variants are distinct and non-overlapping, these two reporters can be co-visualized, and are therefore ideal for in vivo double-labeling or fluorescent energy transfer analyses. The use of GFP and its color variants as reporters provides an unprecedented level of sophistication and represents the next step in mouse genome engineering technology by opening up the possibility of combinatorial non-invasive reporter usage within a single animal.     

Gap junctions or hemichannel-dependent and independent roles of connexins in cataractogenesis and lens development

In the last decade or so, increasing evidences suggest that the mutations of two connexin genes, GJA3 and GJA8, are directly linked to human congenital cataracts in North and Central America, Europe and Asia. GIA3 and GIA8 genes encode gap junction-forming proteins, connexin (Cx) 46 and Cx50, respectively. These two connexins are predominantly expressed in lens fiber cells. Majority of identified mutations are missense, and the mutated sites are scattered across various domains of connexin molecules. Genetic deletion of either of these two genes leads to the development of cataracts; however, the types of cataracts developed are distinctive. More interestingly, microphthalmia is only developed in Cx50, but not Cx46 deficient mice, suggesting the unique role of Cx50 in lens cell growth and development. Knockin studies with the replacement of Cx46 or Cx50 at their respective gene locus further demonstrate the unique properties of these two connexins. Furthermore, the function of Cx50 in epithelial-fiber differentiation appears to be independent of its conventional role in forming gap junction junction channels. Due to their specific functions in maintaining lens clarity and development, and their malfunctions resulting in lens cataractogenesis and developmental impairment, connexin molecules could be developed as potential drug targets for therapeutic intervention for treatment of cataracts and other eye disorders. Recent advances in basic research of lens connexins and the discoveries of clinical disorders as a result of lens connexin dysfunctions are summarized and discussed here.     

Disruption of Gja8 (alpha8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation.

The development of the vertebrate lens utilizes a sophisticated cell-cell communication network via gap junction channels, which are made up of at least three connexin isoforms, alpha8 (Cx50), alpha3 (Cx46) and alpha1 (Cx43), and which are encoded by three different genes. In a previous study, we reported that, with a disruption of Gja3 (alpha3 connexin), mice developed nuclear cataracts with a normal sized lens. We show that Gja8tm1 (alpha8-/-) mice develop microphthalmia with small lenses and nuclear cataracts, while the alpha8 heterozygous (+/-) mice have relatively normal eyes and lenses. A comparative study of these alpha3 and alpha8 knockout mice showed that the protein levels of both alpha3 and alpha8 were independently regulated and there was no compensation for either the alpha3 or alpha8 protein from the wild-type allele when the other allele was disrupted. More interestingly, western blotting data indicated that the presence of alpha8 in the lens nucleus is dependent on alpha3 connexin, but not vice versa. The staining of the knock-in lacZ reporter gene showed the promoter activity of alpha8 connexin is much higher than that of alpha3 connexin in embryonic lenses and in adult lens epithelium. More importantly, a delayed denucleation process was observed in the interior fibers of the alpha8-/- lenses. Therefore, alpha8 connexin is required for proper fiber cell maturation and control of lens size.