Search

Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre-loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies (EBs) without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in EB cultures, representing the inductive key step of further differentiation events. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC-incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx-mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well-described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells.

Functional gap junction channels between neighboring cells can be assessed by microinjection of low molecular weight tracer substances into cultured cells. The extent of direct intercellular communication can be precisely quantified by this method. This protocol describes the iontophoretic injection and visualisation of Neurobiotin into cultured cells.

Distinct mutations in the gap junction protein connexin30 (Cx30) can cause the ectodermal dysplasia Clouston syndrome in humans. We have generated a new mouse line expressing the Clouston syndrome mutation Cx30A88V under the control of the endogenous Cx30 promoter. Our results show that the mutated Cx30A88V protein is incorporated in gap junctional plaques of the epidermis. Homozygous Cx30A88V mice reveal hyperproliferative and enlarged sebaceous glands as well as a mild palmoplantar hyperkeratosis. Additionally, homozygous mutant mice show an altered hearing profile compared to control mice. We conclude that the Cx30A88V mutation triggers hyperproliferation in the skin and changes the cochlear homeostasis in mice.

During repetitive stimulation of skeletal muscle, extracellular ATP levels raise, activating purinergic receptors, increasing Ca2+ influx, and enhancing contractile force, a response called potentiation. We found that ATP appears to be released through pannexin1 hemichannels (Panx1 HCs). Immunocytochemical analyses and function were consistent with pannexin1 localization to T-tubules intercalated with dihydropyridine and ryanodine receptors in slow (soleus) and fast (extensor digitorum longus, EDL) muscles. Isolated myofibers took up ethidium (Etd+) and released small molecules (as ATP) during electrical stimulation. Consistent with two glucose uptake pathways, induced uptake of 2-NBDG, a fluorescent glucose derivative, was decreased by inhibition of HCs or glucose transporter (GLUT4), and blocked by dual blockade. Adult skeletal muscles apparently do not express connexins, making it unlikely that connexin hemichannels contribute to the uptake and release of small molecules. ATP release, Etd+ uptake, and potentiation induced by repetitive electrical stimulation were blocked by HC blockers and did not occur in muscles of pannexin1 knockout mice. MRS2179, a P2Y1R blocker, prevented potentiation in EDL, but not soleus muscles, suggesting that in fast muscles ATP activates P2Y1 but not P2X receptors. Phosphorylation on Ser and Thr residues of pannexin1 was increased during potentiation, possibly mediating HC opening. Opening of Panx1 HCs during repetitive activation allows efflux of ATP, influx of glucose and possibly Ca2+ too, which are required for potentiation of contraction. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Rationale: The gap junctional protein connexin (Cx) 45 is strongly expressed in the early embryonic myocardium. In the adult hearts of mice and humans, the expression mainly is restricted to the cardiac conduction system. Cx45 plays an essential role for development and function of the embryonic heart because general and cardiomyocyte-directed deficiencies of Cx45 in mice lead to embryonic lethality attributable to morphological and functional cardiovascular defects. The function of Cx45 in the adult mouse has not yet been cleared. Objective: To clarify the function of Cx45 in the adult mouse heart. Methods and Results: To circumvent the embryonic lethality resulting from Cx45 deficiency, mice were generated in which deletion of Cx45 specifically was induced in cardiomyocytes of adult mice. These Cx45-deficient mice were viable but showed a decrease in atrioventricular nodal conductivity. In addition, the Cx30.2 protein that is coexpressed with Cx45 in the cardiac conduction system was posttranscriptionally reduced by 70% in mutant hearts. Furthermore, deletion of both Cx45 and Cx30.2 resulted in viable mice that, however, showed stronger impairment of atrioventricular nodal conduction than the single Cx45-deficient mice. Conclusions: Cx45 is required for optimal impulse propagation in the atrioventricular node and stabilizes the level of the coexpressed Cx30.2 protein in the adult mouse heart. In contrast to the embryo, Cx45 is not essential for the viability of adult mice.

The gap junction channel protein connexin40 (Cx40) is crucial in vascular and renal physiology, because Cx40-deficient mice exhibit impaired conduction of endothelium-dependent dilations and pronounced hypertension. The latter precludes mechanistic insights into the role of endothelial Cx40, because long-lasting hypertension itself may affect conduction and Cx expression. We aimed to identify endothelial Cx40 functions, their dependency on the conductive capability, and to separate these from hypertension-related alterations. We assessed conduction and Cx expression in mice with cell type–specific deletion of Cx40 and in mice expressing a defective Cx40 (Cx40A96S) identified in humans, which forms nonconducting gap junction channels. Confined arteriolar stimulation with acetylcholine or bradykinin elicited local dilations that conducted upstream without attenuation of the amplitude for distances up to 1.2-mm in controls with a floxed Cx40 gene (Cx40 fl/fl ). Conducted responses in hypertensive animals devoid of Cx40 in renin-producing cells were unaltered but remote dilations were reduced in normotensive animals deficient for Cx40 in endothelial cells (Cx40 fl/fl :Tie2-Cre). Surprisingly, Cx37 expression was undetectable by immunostaining in arteriolar endothelium only in Cx40 fl/fl :Tie2-Cre; however, transcriptional activity of Cx37 in the cremaster was comparable with Cx40 fl/fl controls. Cx40A96S mice were hypertensive with preserved expression of Cx40 and Cx37. Nevertheless, conducted responses were blunted. We conclude that endothelial Cx40 is necessary to support conducted dilations initiated by endothelial agonists and to locate Cx37 into the plasma membrane. These functions are unaltered by long-lasting hypertension. In the presence of a nonconducting Cx40, Cx37 is present but cannot support the conduction highlighting the importance of endothelial Cx40.

Connexin (Cx) proteins localized to neuronal and glial syncytia provide the ultrastructural components for intercellular communication via gap junctions. In this study, a Cx45 reporter mouse model in which the Cx45 coding sequence is substituted for enhanced green fluorescent protein (eGFP) was used to characterize Cx45 expressing neurones within adult mouse spinal cord. eGFP-immunoreactive (eGFP-IR) cells were localized at all rostro-caudal levels to laminae I–III of the dorsal horn (DH), areas associated with nociception. The neuronal rather than glial phenotype of these cells in DH was confirmed by co-localisation of eGFP-IR with the neuronal marker NeuN. Further immunohistochemical studies revealed that eGFP-IR interneurones co-express the calcium-binding protein calbindin, and to a lesser extent calretinin. In contrast, eGFP-IR profiles did not co-localize with either parvalbumin or GAD-67, both of which are linked to inhibitory interneurones. Staining with the primary afferent markers isolectin-B4 (IB4) and calcitonin gene-related peptide revealed that eGFP-IR somata within laminae I–III receive close appositions from the former, presumed non-peptidergic nociceptive afferents of peripheral origin. The presence of 5-HT terminals in close apposition to eGFP-IR interneuronal somata suggests modulation via descending pathways. These data demonstrate a highly localized expression of Cx45 in a population of interneurones within the mouse superficial dorsal horn. The implications of these data in the context of the putative role of Cx45 and gap junctions in spinal somatosensory processing and pain are discussed.

Expression of connexin26 (Cx26), Cx30 and Cx43 in astrocytes and expression of Cx29, Cx32 and Cx47 in oligodendrocytes of adult rodent brain has been well documented, as has the interdependence of connexin expression patterns of macroglial cells in Cx32- and Cx47-knockout mice. To investigate this interdependence further, we examined immunofluorescence labelling of glial connexins in transgenic Cx30 null mice. Ablation of astrocytic Cx30, confirmed by the absence of immunolabelling for this connexin in all brain regions, resulted in the loss of its coupling partner Cx32 on the oligodendrocyte side of astrocyte-oligodendrocyte (A/O) gap junctions, but had no effect on the localization of astrocytic Cx43 and oligodendrocytic Cx47 at these junctions or on the distribution of Cx32 along myelinated fibres. Surprisingly, gene deletion of Cx30 led to the near total elimination of immunofluorescence labelling for Cx26 in all leptomeningeal tissues covering brain surfaces as well as in astrocytes of brain parenchyma. Moreover northern blot analysis revealed downregulation of Cx26 mRNA in Cx30-knockout brains. Our results support earlier observations on the interdependency of Cx30/Cx32 targeting to A/O gap junctions and further suggest that Cx26 mRNA expression is affected by Cx30 gene expression. In addition, Cx30 protein may be required for co-stabilization of gap junctions or for co-trafficking in cells.

Astrocytes are known to express the gap junction forming proteins connexin30 (Cx30) and connexin43 (Cx43), but it has remained controversial whether these cells also express connexin26 (Cx26). To investigate this issue further, we examined immunofluorescence labelling of glial connexins in wild-type vs. transgenic mice with targeted deletion of Cx26 in neuronal and glial cells (Cx26fl/fl:Nestin-Cre mice). The Cx26 antibodies utilized specifically recognized Cx26 and lacked cross reaction with highly homologous Cx30, as demonstrated by immunoblotting and immunofluorescence in Cx26-transfected and Cx30-transfected C6 glioma cells. Punctate immunolabelling of Cx26 with these antibodies was observed in leptomeninges and subcortical brain regions. This labelling was absent in subcortical areas of Cx26fl/fl:Nestin-Cre mice, but persisted in leptomeningeal tissues of these mice, thereby distinguishing localization of Cx26 between parenchymal and non-parenchymal tissue. In subcortical brain parenchyma, Cx26-positive puncta were often co-localized with astrocytic Cx43, and some were localized along astrocyte cell bodies and processes immunolabelled for glial fibrillary acidic protein. Cx26-positive puncta were also co-localized with punctate labelling of Cx47 around oligodendrocyte somata. Comparisons of Cx26 labelling in rodent species revealed a lower density of Cx26-positive puncta and a more restricted distribution in subcortical regions of mouse compared with rat brain, perhaps partly explaining reported difficulties in detection of Cx26 in mouse brain parenchyma using antibodies or Cx26 gene reporters. These results support our earlier observations of Cx26 expression in astrocytes and its ultrastructural localization in individual gap junction plaques formed between astrocytes as well as in heterotypic gap junctions between astrocytes and oligodendrocytes.

Gap junctional coupling of renin-producing cells is of major functional importance for the control of renin synthesis and release. This study was designed to determine the relevance of the vascular gap junction protein connexin 45 (Cx45) for the control of renin expression and secretion. By crossbreeding mice which drive Cre recombinase under the control of the endogenous renin promoter with mice harboring floxed Cx45 gene alleles, we generated viable mice with a deletion of Cx45 in the renin cell lineage. These mice were normotensive, and renin cells in their kidneys were normal with regard to localization and number. Sodium deficiency induced typical recruitment of renin-producing cells along afferent arterioles, whereas sodium overload resulted in a decrease in the number of cells expressing renin. Regulation of renin secretion by perfusion pressure, catecholamines, and angiotensin II from isolated kidneys of mice with renin cell-specific deletion of Cx45 was normal. Analyzing Cx45 promoter activity in cells of the preglomerular arteriolar tree by using mice driving the reporter gene LacZ under the control of the Cx45 promoter revealed strong staining in smooth muscle cells of the media, whereas renin-expressing cells were almost devoid of LacZ staining. Conversely, renin-producing cells, but not vascular smooth muscle cells expressed the gap junction protein Cx40. These findings suggest that Cx45 plays no major functional role in renin-producing cells, probably because the expression of Cx45 is downregulated in these cells. Since renin-producing cells in the adult kidney can reversibly transform into vascular smooth muscle cells and vice versa, our findings on connexin expression indicate that these phenotype switches are paralleled by characteristic reciprocal changes in the transcriptional activity of Cx40 and Cx45 genes.

In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild-type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45-EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons. J. Comp. Neurol. 519:433–450, 2011. © 2010 Wiley-Liss, Inc.

The gap junction protein connexin-45 (Cx45) is expressed in the conduction system of the heart and in certain neurons of the retina and brain. General and cardiomyocyte-directed deficiencies of Cx45 in mice lead to lethality on embryonic day 10.5 as a result of cardiovascular defects. Neuron-directed deletion of Cx45 leads to defects in transmission of visual signals. Connexin-36 (Cx36) is co-expressed with Cx45 in certain types of retinal interneurons. To determine whether these two connexins have similar functions and whether Cx36 can compensate for Cx45, we generated knock-in mice in which DNA encoding Cx45 was replaced with that encoding Cx36. Neuron-directed replacement of Cx45 with Cx36 resulted in viable animals. Electroretinographic and neurotransmitter coupling analyses demonstrated functional compensation in the retina. By contrast, general and cardiomyocyte-directed gene replacement led to lethality on embryonic day 11.5. Mutant embryos displayed defects in cardiac morphogenesis and conduction. Thus, functional compensation of Cx45 by Cx36 did not occur during embryonic heart development. These data suggest that Cx45 and Cx36 have similar functions in the retina, whereas Cx45 fulfills special functions in the developing heart that cannot be compensated by Cx36.

Arterial PCO2, a major determinant of breathing, is detected by chemosensors located in the brainstem. These are important for maintaining physiological levels of PCO2 in the blood and brain, yet the mechanisms by which the brain senses CO(2) remain controversial. As ATP release at the ventral surface of the brainstem has been causally linked to the adaptive changes in ventilation in response to hypercapnia, we have studied the mechanisms of CO(2)-dependent ATP release in slices containing the ventral surface of the medulla oblongata. We found that CO(2)-dependent ATP release occurs in the absence of extracellular acidification and correlates directly with the level of PCO2. ATP release is independent of extracellular Ca(2+) and may occur via the opening of a gap junction hemichannel. As agents that act on connexin channels block this release, but compounds selective for pannexin-1 have no effect, we conclude that a connexin hemichannel is involved in CO(2)-dependent ATP release. We have used molecular, genetic and immunocytochemical techniques to demonstrate that in the medulla oblongata connexin 26 (Cx26) is preferentially expressed near the ventral surface. The leptomeninges, subpial astrocytes and astrocytes ensheathing penetrating blood vessels at the ventral surface of the medulla can be loaded with dye in a CO(2)-dependent manner, suggesting that gating of a hemichannel is involved in ATP release. This distribution of CO(2)-dependent dye loading closely mirrors that of Cx26 expression and colocalizes to glial fibrillary acidic protein (GFAP)-positive cells. In vivo, blockers with selectivity for Cx26 reduce hypercapnia-evoked ATP release and the consequent adaptive enhancement of breathing. We therefore propose that Cx26-mediated release of ATP in response to changes in PCO2 is an important mechanism contributing to central respiratory chemosensitivity.

Mutations in the GJB2 and GJB6 genes, respectively, coding for connexin26 (Cx26) and connexin30 (Cx30) proteins, are the most common cause for prelingual non-syndromic deafness in humans. In the inner ear, Cx26 and Cx30 are expressed in different non-sensory cell types, where they largely co-localize and may form heteromeric gap junction channels. Here, we describe the generation and characterization of a mouse model for human bilateral middle/high-frequency hearing loss based on the substitution of an evolutionarily conserved threonine by a methionine residue at position 5 near the N-terminus of Cx30 (Cx30T5M). The mutation was inserted in the mouse genome by homologous recombination in mouse embryonic stem cells. Expression of the mutated Cx30T5M protein in these transgenic mice is under the control of the endogenous Cx30 promoter and was analysed via activation of the lacZ reporter gene. When probed by auditory brainstem recordings, Cx30(T5M/T5M) mice exhibited a mild, but significant increase in their hearing thresholds of about 15 dB at all frequencies. Immunolabelling with antibodies to Cx26 or Cx30 suggested normal location of these proteins in the adult inner ear, but western blot analysis showed significantly down-regulated the expression levels of Cx26 and Cx30. In the developing cochlea, electrical coupling, probed by dual patch-clamp recordings, was normal. However, transfer of the fluorescent tracer calcein between cochlear non-sensory cells was reduced, as was intercellular Ca(2+) signalling due to spontaneous ATP release from connexin hemichannels. Our findings link hearing loss to decreased biochemical coupling due to the point-mutated Cx30 in mice.

Mouse connexin57 (Cx57) is expressed most abundantly in horizontal cells of the retina, and forms gap junction (GJ) channels, which constitute a structural basis for electrical and metabolic intercellular communication, and unapposed hemichannels (UHCs) that are involved in an exchange of ions and metabolites between the cytoplasm and extracellular milieu. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we showed that HeLa cells expressing Cx57 and C-terminally fused with enhanced green fluorescent protein (Cx57-EGFP) form junctional plaques (JPs) and that only cell pairs exhibiting at least one JP demonstrate cell-to-cell electrical coupling and transfer of negatively and positively charged dyes with molecular mass up to ∼400 Da. The permeability of the single Cx57 GJ channel to Alexa fluor-350 is ∼90-fold smaller than the permeability of Cx43, while its single channel conductance (57 pS) is only 2-fold smaller than Cx43 (110 pS). Gating of Cx57-EGFP/Cx45 heterotypic GJ channels reveal that Cx57 exhibit a negative gating polarity, i.e. channels tend to close at negativity on the cytoplasmic side of Cx57. Alkalization of pHi from 7.2 to 7.8 increased gap junctional conductance (gj) of ∼100-fold with pKa= 7.41. We show that this gj increase was caused by an increase of both the open channel probability and the number of functional channels. Function of Cx57 UHCs was evaluated based on the uptake of fluorescent dyes. We found that under control conditions, Cx57 UHCs are closed and open at [Ca2+]o=∼0.3 mm or below, demonstrating that a moderate reduction of [Ca2+]o can facilitate the opening of Cx57 UHCs. This was potentiated with intracellular alkalization. In summary, our data show that the open channel probability of Cx57 GJs can be modulated by pHi with very high efficiency in the physiologically relevant range and may explain pH-dependent regulation of cell–cell coupling in horizontal cell in the retina.

In the human heart connexin(Cx)40, Cx43 and Cx45-containing gap junctional channels electrically couple cardiomyocytes, forming a functional syncytium. In the mouse heart, additionally, Cx30.2-containing gap junctions have been detected in the atrioventricular node where they are implicated, together with Cx45, in impulse delay. However, whether the human ortholog of Cx30.2, Cx31.9, is expressed in the human heart has not previously been investigated. We therefore generated Cx31.9 specific antibodies to test for the expression of Cx31.9 in the human heart. These antibodies recognized the Cx31.9 protein in HeLaCx31.9 transfectants by immunofluorescence and immunoblot analyses. However, we did not find punctate Cx31.9 specific immunofluorescence signals in the working myocardium or in the impulse generation and conduction system of adult or fetal human heart. Complementary immunoblot analyses did not reveal Cx31.9 protein in the adult atrial or ventricular myocardium. We conclude that the Cx31.9 protein, unlike its counterpart in the mouse, is not expressed in detectable quantities and is thus unlikely to contribute to the impulse generation and conduction system or the working myocardium of the human heart.

In the adult dentate gyrus, radial glia-like cells represent putative stem cells generating neurons and glial cells. Here, we combined patch-clamp recordings, biocytin filling, immunohistochemistry, single-cell transcript analysis, and mouse transgenics to test for connexin expression and gap junctional coupling of radial glia-like cells and its impact on neurogenesis. Radial glia-like cells were identified in mice expressing EGFP under control of the nestin and gfap promoters. We show that a majority of Radial glia-like cells are coupled and express Cx43. Neuronal precursors were not coupled. Mice lacking Cx30 and Cx43 in GFAP-positive cells displayed almost complete inhibition of proliferation and a significant decline in numbers of radial glia-like cells and granule neurons. Inducible virus-mediated ablation of connexins in the adult hippocampus also reduced neurogenesis. These findings strongly suggest the requirement of connexin expression by radial glia-like cells for intact neurogenesis in the adult brain and point to possible communication pathways of these cells.

Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed β-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the propagation of JGA vascular signals and in the regulation of renin release and blood pressure.

Preconditioning is an endogenous mechanism in which a nonlethal exposure increases cellular resistance to subsequent additional severe injury. Here we show that connexin 43 (Cx43) plays a key role in protection afforded by preconditioning. Cx43 null mice were insensitive to hypoxic preconditioning, whereas wild-type littermate mice exhibited a significant reduction in infarct volume after occlusion of the middle cerebral artery. In cultures, Cx43-deficient cells responded to preconditioning only after exogenous expression of Cx43, and protection was attenuated by small interference RNA or by channel blockers. Our observations indicate that preconditioning reduced degradation of Cx43, resulting in a marked increase in the number of plasma membrane Cx43 hemichannels. Consequently, efflux of ATP through hemichannels led to accumulation of its catabolic product adenosine, a potent neuroprotective agent. Thus, adaptive modulation of Cx43 can offset environmental stress by adenosine-mediated elevation of cellular resistance.

Haplodeficient mice expressing carboxyl-terminally truncated Cx43 (K258stop/KO), instead of the wild-type Cx43 isoform, reach adulthood and reveal no abnormalities in heart morphology. Here, we have analyzed the expression of K258stop protein and the morphology of gap junctions in adult hearts of these mice. Coimmunofluorescence analysis revealed reduced juxtaposition of K258stop with other junctional proteins at the intercalated disc. Immunoprecipitation studies documented changes in the interaction with previously described Cx43 binding proteins. Quantitative transmission electron and confocal microscopy confirmed the localization of K258stop gap junctions to the periphery of the intercalated disc and further revealed an increase in the size of K258stop gap junction plaques and a reduction in their number. Dual whole cell patch clamp analysis confirmed that K258stop gap junctions were functional, with single channel properties similar to those described in exogenous systems. We conclude that the carboxyl-terminal domain of Cx43 (Cx43CT) is involved in regulating the localization, number and size of Cx43 plaques in vivo. Conversely, protein interactions or posttranslational modifications taking place within the Cx43CT are not required for the assembly of functional gap junctions in the intercalated disc.

The gap junction gene Connexin31.1 has been reported to be expressed predominantly in the epidermis of murine skin. To study the function of this gene, we generated mice in which the coding DNA of the Connexin31.1 gene was replaced by lacZ reporter coding DNA. Using β-galactosidase staining, we have shown that lacZ/Connexin31.1 was expressed in the spinous and granular layers of the epidermis, in cells of olfactory epithelium and in the vomeronasal organ. During embryogenesis, Connexin31.1 was co-expressed with another isoform, Connexin31, in the post-implantation trophoblast cell lineage and, later in gestation, in placental glycogen cells. Although homozygous Connexin31.1-deficient mice were fertile and showed no morphological or functional defects in adult organs expressing this gene, 30% of the offspring expected according to Mendelian inheritance were lost between embryonic days 11.5 and 14.5 and surviving embryos were significantly reduced in weight near the end of pregnancy. Placentas of Connexin31.1-deficient embryos were reduced in weight and showed altered morphology of the spongiotrophoblast and labyrinth layer. The spongiotrophoblast formed a compact barrier at the decidual border that might restrict the maternal blood supply. We conclude that Connexin31.1 is critical for normal placental development but appears to be functionally compensated by other connexin isoforms in the embryo proper and adult mouse.

Erythrokeratodermia variabilis (EKV) is a rare autosomal dominant human genodermatosis. Its clinical appearance varies from transient, fast moving erythemas to persistent brown hyperkeratoses. So far, several mutations in the Cx31 or Cx30.3 gene have been reported to cause EKV in humans. We have generated a conditional mouse mutant that carries the human F137L mutation in the Cx31 gene which was described to act in a transdominant negative manner. The phenylalanine residue at position 137 is highly conserved in several human and mouse connexin genes. Mouse embryonic stem (ES) cells expressing one allele of the Cx31F137L mutation were stable but showed approximately 30% decreased transfer of neurobiotin. This is probably due to dominant negative effects of the Cx31F137L protein on wild type Cx31 and Cx43 protein expressed in ES cells. Surprisingly, the healing process of tail incision wounds in Cx31(+/F137L) mice was shortened by 1 day, i.e. very similar as previously reported for mice with decreased expression of Cx43 in the epidermis. This suggests again that Cx31 and Cx43 proteins functionally interact, possibly by forming heteromeric channels in the epidermis. Heterozygous Cx31(+/F137L) mice are viable and fertile, in contrast to homozygous Cx31(F137L/F137L) mice that die around ED 7.5. In Cx31(+/F137L) mice, the epidermal expression pattern and level of Cx26, Cx30, Cx30.3 and Cx43 proteins were not altered compared with wild-type mice. No erythemas were detected in young C31(+/F137L) mice before 2 weeks of age. In contrast to human EKV patients, hyperproliferation of the stratum germinativum was found in only 5% of the analyzed skin area.

Mutations in connexin (Cx) genes are responsible for a large proportion of human inherited prelingual deafness cases. The most commonly found human Cx mutations are either Cx26 or Cx30 deletions. Histological observations made in the organ of Corti of homozygous Cx26 and Cx30 gene knockout mice show that cochlear hair cells degenerate after the onset of hearing. However, it is unclear whether vestibular hair cells undergo similar degeneration in connexin knockout mice. To address this question, we first examined expression patterns of Cx26 and Cx30 in the saccule, utricle, and ampulla by immunolabeling. In wild-type mice, Cx26 and Cx30 immunoreactivity was found extensively in vestibular supporting cells and connective tissue cells, and the two Cxs were co-localized in most gap junction (GJ) plaques. Targeted deletion of the Cx30 gene, which caused little change in Cx26 expression pattern, resulted in a significant and age-related loss of vestibular hair cells only in the saccule. dUTP nick end labeling (TUNEL) staining also revealed on-going apoptosis specifically in saccular hair cells of Cx30(-/-) mice. These results indicated that hair cell survival in the utricle and ampulae does not require Cx30. Importantly, over-expressing the Cx26 gene from a modified bacterial artificial chromosome in the Cx30(-/-) background rescued the saccular hair cells. These results suggest that it is the reduction in the total amount of GJs rather than the specific loss of Cx30 that underlies saccular hair cell death in Cx30(-/-) mice. Hybrid GJs co-assembled from Cx26 and Cx30 were not essential for the survival of saccular hair cells.

The keratitis-ichthyosis-deafness (KID) syndrome is characterized by corneal, skin, and hearing abnormalities. KID has been linked to heterozygous dominant missense mutations in the GJB2 and GJB6 genes, encoding connexin26 and 30, respectively. In vitro evidence indicates that KID mutations lead to hyperactive (open) hemichannels, which in some cases is accompanied by abnormal function of gap junction channels. Transgenic mouse models expressing connexin26 KID mutations reproduce human phenotypes and present impaired epidermal calcium homeostasis and abnormal lipid composition of the stratum corneum affecting the water barrier. Here we have compiled relevant data regarding the KID syndrome and propose a mechanism for the epidermal aspects of the disease.

Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) show numerous alterations including inflammation, apoptosis, and necrosis of myofibers. However, the molecular mechanism that explains these changes remains largely unknown. Here, the involvement of hemichannels formed by connexins (Cx HCs) was evaluated in skeletal muscle of mdx mouse model of DMD. Fast myofibers of mdx mice were found to express three connexins (39, 43 and 45) and high sarcolemma permeability, which was absent in myofibers of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca(2+) levels, immunoreactivity to phosphorylated p65 (active NF-κB), eNOS and annexin V/active Caspase 3 (marker of apoptosis) but presented dystrophin immunoreactivity. Moreover, muscles of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice exhibited partial decrease of necrotic features (big cells and high creatine kinase levels). Accordingly, these muscles showed similar macrophage infiltration as control mdx muscles. Nonetheless, the hanging test performance of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice was significantly better than that of control mdx Cx43(fl/fl)Cx45(fl/fl) mice. All three Cxs found in skeletal muscles of mdx mice were also detected in fast myofibers of biopsy specimens from patients with muscular dystrophy. Thus, reduction of Cx expression and/or function of Cx HCs may be potential therapeutic approaches to abrogate myofiber apoptosis in DMD.

The keratitis–ichthyosis–deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome.

In mouse heart, four connexins (Cxs), Cx30.2, Cx40, Cx43, and Cx45, form gap junction (GJ) channels for electric and metabolic cell-to-cell signaling. Extent and pattern of Cx isoform expression together with cytoarchitecture and excitability of cells determine the velocity of excitation spread in different regions of the heart. In the SA node, cell-cell coupling is mediated by Cx30.2 and Cx45, which form low-conductance (approximately 9 and 32 pS, respectively) GJ channels. In contrast, the working cardiomyocytes of atria and ventricles express mainly Cx40 and Cx43, which form GJ channels of high conductance (approximately 180 and 115 pS, respectively) that facilitate the fast conduction necessary for efficient mechanical contraction. In the AV node, cell-cell coupling is mediated by abundantly expressed Cx30.2 and Cx45 and Cx40, which is expressed to a lesser extent. Cx30.2 and Cx45 may determine higher intercellular resistance and slower conduction in the SA- and AV-nodal regions than in the ventricular conduction system or the atrial and ventricular working myocardium. Cx30.2 and its putative human ortholog, Cx31.9, under physiologic conditions form unapposed hemichannels in nonjunctional plasma membrane; these hemichannels have a conductance of approximately 20 pS and are permeable to cationic dyes up to approximately 400 Da in molecular mass. Genetic ablation of Cxs confirmed that Cx40 and Cx43 are important in determining the high conduction velocities in atria and ventricles, whereas the deletion of the Cx30.2 complementary DNA led to accelerated conduction in the AV node and reduced the Wenckebach period. We suggest that these effects are caused by (1) a dominant-negative effect of Cx30.2 on junctional conductance via formation of low-conductance homotypic and heterotypic GJ channels, and (2) open Cx30.2 hemichannels in non-junctional membranes, which shorten the space constant and depolarize the excitable membrane.

In the gastrulating mouse embryo, the gap junction protein connexin43 is expressed exclusively in cells derived from the inner cell mass, whereas connexin31 is expressed in cells of the trophoblast lineage. Since connexin43 and connexin31 do not form heterotypic gap junction channels in exogenous expression systems, such as HeLa cells and Xenopus oocytes, previous studies have suggested that the incompatibility of these two connexins could contribute to the separation of connexin43-expressing and connexin31-expressing compartments between embryo and extraembryonic tissues at gastrulation, respectively. Thus, we have generated connexin43 knock-in connexin31 mice, in which the coding region of the connexin43 gene was replaced by that of connexin31. Interbreeding of heterozygous connexin43 knock-in connexin31 mice resulted in homozygous connexin43 knock-in connexin31 mice, but none of them survived to adulthood. As these mice were born at the expected Mendelian frequency, we conclude that the reported incompatibility of connexin43 and connexin31 to form heterotypic gap junction channels does not interfere with normal embryonic development. Neonatal homozygous connexin43 knock-in connexin31 hearts showed malformation in the subpulmonary outlet of the right ventricle, similar to general connexin43-deficient mice. Electrocardiograms of neonatal hearts in homozygous connexin43 knock-in connexin31 mice revealed significantly low voltage of the QRS complex. This is in contrast to previous results from our laboratory which showed that replacement of connexin43 by connexin40 resulted in morphologically and functionally normal hearts. We conclude that connexin31 cannot functionally replace connexin43 during cardiac morphogenesis.

Recent research results indicate that glial gap-junction communication is much more complex and widespread than originally thought, and has diverse roles in brain homeostasis and the response of the brain to injury. The situation is far from clear, however. Pharmacological agents that block gap junctions can abolish neuron-glia long-range signaling and can alleviate neuronal damage whereas, intriguingly, opposite effects are observed in mice lacking connexin43, a major gap-junction subunit protein in astrocytes. How can the apparently contradictory results be explained, and how is specificity achieved within the glial gap-junction system? Another key issue in understanding glial connexin function is that oligodendrocytes and astrocytes, each of which express distinct connexin isotypes, are thought to participate in brain homeostasis by forming a panglial syncytium. Molecular analysis has revealed a surprising diversity of connexin expression and function, and this has led to new hypotheses regarding their roles in the brain, which could be tested using new approaches.

Previous studies have shown that two subpopulations of cells with astrocytic properties coexist in the mouse hippocampus, which display distinct morpho- logical and functional characteristics, specifically a nonoverlapping expression of either AMPA-type glutamate receptors (GluR cells) or glutamate transporters (GluT cells). Use of transgenic mice with hGFAP promoter-controlled EGFP expression and patch-clamp recordings allow reliable identification of the two cell types in hippocampal slices. Extending functional characterization, we report here the complete lack of gap junc- tional tracer coupling in GluR cells, while GluT cells are shown to be extensively coupled. This distinction is valid in immature as well as adult animals. Analysis of transgenic mice expressing -Gal under regulatory elements of the Cx43 promoter revealed the absence of Cx43 in GluR cells. Experiments using gap junction blockers demonstrated that passive currents, displayed primarily by GluT cells, do not reflect intercellular coupling but are attributable to intrinsic membrane properties of individ- ual cells. This study supports the notion that the two subpopulations of hGFAP-EGFP- positive cells represent distinct cell types with contrasting physiological properties. Since GluR cells do not participate in the astrocytic gap junctional network, their functional role must be different from spatial buffering of ions or signaling molecules, i.e., properties generally assigned to astrocytes. © 2004 Wiley-Liss, Inc.

Intercellular communication through gap junctions is crucial for proper functioning of the inner ear. Indeed, mutations in several connexin genes have been found to cause hearing loss. In the inner ear, only the cell distributions of connexin30 and connexin26 have been well documented. We took advantage of the lacZ reporter gene in Cx43 and Cx45 knock-out mice to study the expression of the connexin43 and connexin45 genes during the inner ear development. Expression of Cx43 and Cx45 in the inner ear was detected from embryonic days 15.5 and 17.5, respectively. Until the 1st week of life, Cx43 was highly expressed in the connective tissues, and weakly expressed in the immature sensory epithelium of the cochlea. From postnatal day 8, however, Cx43 was almost exclusively expressed in the bone of the otic capsule. During embryogenesis, Cx45 was expressed in epithelial and connective inner ear tissues. From birth onwards, Cx45 expression could be detected in some inner ear capillaries. Vascular expression thereafter increased and persisted in the adult. In the mature inner ear, Cx45 was expressed in the entire vasculature. These results indicate that connexin43 and connexin45 play a role in the otic capsule bone and the inner ear vascular system, respectively.

To analyze the effect of connexin loss on the repair of wounded tail skin, we have studied the following transgenic mouse mutants: connexin30 –/– , connexin31 –/– and connexin43 Cre-ER(T)/fl (for inducible deletion of the connexin43 coding region). Connexin43 and connexin31 are expressed in the basal and spinous layers of wild-type epidermis, whereas connexin31 and small amounts of connexin30, as well as connexin26 proteins, were found in the granulous layer. Connexin43 was downregulated in connexin31 -deficient mice, whereas mice with reduced connexin43 exhibited an upregulation of connexin30. During wound healing, connexin30 and connexin26 proteins were upregulated in all epidermal layers, whereas connexin43 and connexin31 protein expression were downregulated. In connexin31 –/– mice, reduced levels of connexin30 protein were observed on days 1 and 2 after wounding. The closure of epidermal wounds in mice with decreased amounts of connexin43 protein occurred one day earlier. Under these conditions the expression profiles of connexin30 and connexin31 were also temporarily shifted by one day. Furthermore, dye transfer between keratinocytes in skin sections from connexin43 -deficient mice was decreased by 40%. These results suggest that downregulation of connexin43 appears to be a prerequisite for the coordinated proliferation and mobilization of keratinocytes during wound healing.

Characterization of the expression pattern of connexins in neural tissue is a necessary prerequisite for understanding the functional relevance of the corresponding gap junction channels in brain. Here we describe the cell type-specific expression of connexin45 in the CNS and the spatiotemporal expression pattern from embryonic day 19.5 to adult brain using a recently described connexin45 LacZ-reporter mouse. The connexin45 gene is highly expressed during embryogenesis and up to 2 weeks after birth in nearly all brain regions. Afterward its expression is restricted to the thalamus, the CA3 region of hippocampus and the cerebellum. In adult mouse brain, the pattern of LacZ-staining in combination with the analysis of different neuronal and glial marker proteins strongly suggests that connexin45 is expressed in neurons, but presumably not in astrocytes or mature oligodendrocytes. Expression of the LacZ/connexin45 reporter gene in subsets of neurons, such as cerebral cortical, hippocampal and thalamic neurons as well as basket and stellate cells of cerebellum should be corroborated by functional investigations of connexin45 protein in electrical synapses. Based on its expression pattern during development, we suggest that the connexin45-containing gap junction channels have a rather ubiquitous role during brain development and may contribute to functional specification in certain subsets of neurons in the adult brain.

Compensatory mechanisms after genetic manipulations have been documented extensively for the nervous system. In many cases, these mechanisms involve genetic regulation at the transcription or expression level of existing isoforms. We report a novel mechanism by which single neurons compensate for changes in network connectivity by retuning their intrinsic electrical properties. We demonstrate this mechanism in the inferior olive, in which widespread electrical coupling is mediated by abundant gap junctions formed by connexin 36 (Cx36). It has been shown in various mammals that this electrical coupling supports the generation of subthreshold oscillations, but recent work revealed that rhythmic activity is sustained in knock-outs of Cx36. Thus, these results raise the question of whether the olivary oscillations in Cx36 knock-outs simply reflect the status of wild-type neurons without gap junctions or the outcome of compensatory mechanisms. Here, we demonstrate that the absence of Cx36 results in thicker dendrites with gap-junction-like structures with an abnormally wide interneuronal gap that prevents electrotonic coupling. The mutant olivary neurons show unusual voltage-dependent oscillations and an increased excitability that is attributable to a combined decrease in leak conductance and an increase in voltage-dependent calcium conductance. Using dynamic-clamp techniques, we demonstrated that these changes are sufficient to transform a wild-type neuron into a knock-out-like neuron. We conclude that the absence of Cx36 in the inferior olive is not compensated by the formation of other gap-junction channels but instead by changes in the cytological and electroresponsive properties of its neurons, such that the capability to produce rhythmic activity is maintained.

To further characterize the recently described gap junction gene connexin 47 (Cx47), we generated Cx47-null mice by replacing the Cx47 coding DNA with an enhanced green fluorescent protein (EGFP) reporter gene, which was thus placed under control of the endogenous Cx47 promoter. Homozygous mutant mice were fertile and showed no obvious morphological or behavioral abnormalities. Colocalization of EGFP fluorescence and immunofluorescence of cell marker proteins revealed that Cx47 was mainly expressed in oligodendrocytes in highly myelinated CNS tissues and in few calcium-binding protein S100β subunit-positive cells but not in neurons or peripheral sciatic nerve. This corrects our previous conclusion that Cx47 mRNA is expressed in brain and spinal cord neurons (Teubner et al., 2001). Cx47 protein was detected by Western blot analysis after immunoprecipitation in CNS tissues of wild-type mice but not in heart or Cx47-deficient tissues. Electron microscopic analysis of CNS white matter in Cx47-deficient mice revealed a conspicuous vacuolation of nerve fibers, particularly at the site of the optic nerve where axons are first contacted by oligodendrocytes and myelination starts. Initial analyses of Cx32/Cx47-double-deficient mice showed that these mice developed an action tremor and died on average at 51 d after birth. The central white matter of these double-deficient mice exhibited much more abundant vacuolation in nerve fibers than mice deficient only in Cx47.

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.

Recent evidence suggests that electrotonic coupling is an important mechanism for neuronal synchronisation in the mammalian cortex and hippocampus. Various types of network oscillations have been shown to depend on, or be sharpened by, gap junctions between inhibitory interneurones or excitatory projection cells. Here we made use of a targeted disruption of the gene coding for Cx36, a recently discovered neuronal gap junction subunit, to analyse its role in hippocampal network behaviour. Mice lacking Cx36 are viable and lack obvious morphological or behavioural abnormalities. Stimulation of afferent and efferent fibre pathways in hippocampal slices revealed a largely normal function of the synaptic circuitry, including tetanically evoked network oscillations. Spontaneous sharp waves and ripple (approximately 200 Hz) oscillations, however, occurred less frequently in slices from Cx36 -/- mice, and ripples were slightly slower than in littermate controls. Moreover, epileptiform discharges elicited by 4-aminopyridine were attenuated in slices from Cx36 -/- mice. Our findings indicate that Cx36 plays a role in the generation of certain forms of network synchronisation in the hippocampus, namely sharp wave-ripple complexes and hypersynchronous epileptiform discharges.

Communication and interaction between cells has been shown to be important during the embryonic development of the vertebrate hindbrain, which becomes transiently subdivided into segments called rhombomeres (r). One gene family allowing intercellular communication and possibly being involved in the control of hindbrain development is the connexin family encoding gap junction channels. Here, we have characterized in detail the previously observed (Dahl et al., 1997) expression of one particular connexin gene, connexin31 (Cx31), in the mouse embryonic hindbrain and compared it with that of Cx43 and Cx36. We found transient Cx31 expression from approximately embryonic day (E) E8-E11 in two small lateral/dorsal subgroups of cells in the hindbrain. We could show that these spots of expression corresponded to r3 and r5 and that Cx31 expression in r3 and r5 was controlled by the transcription factor Krox20. In contrast, expression of Cx43 and Cx36 started later (from E9.5 and E10.5, respectively) and was confined to longitudinal stripes of expression. In addition, from E10.5-E11.5, Cx31 was expressed by a column of cells in ventral r4, most likely representing contralateral vestibulo-acoustic efferent neurons, immediately anterior to a ventral column expressing Cx36 at the same stage. From E11.5 onward, another site of Cx31 expression was detected in the boundary cap cells in the entry/exit points of all mixed sensory/motor and in the entry points of pure sensory nerves. This expression was not present in the boundary cap cells of the exit points of pure motor nerves. So far, our analysis of the hindbrain area of Cx31-deficient embryos in terms of projections of sensory or motor neurons or in the generation or migration of neurons has not yet revealed any obvious defects.

In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.

Connexins are the protein subunits of gap junction channels and are expressed in a highly regulated temporal and spatial pattern in embryonic development and adult life, with most cell types expressing more than one isoform. Connexin31 (Cx31) is encoded by the gene Gjb3 and expressed throughout mouse development n a complex pattern; in adult mice it becomes restricted to the granular layer of epidermis, testis, and placenta. In placenta, lack of Cx31 leads to transient dysmorphogenesis affecting embryonic survival. Here we have analyzed the structure of mouse Gjb3 as well as its transcriptional regulation by transient transfection of reporter gene constructs in HM1 mouse embryonic stem cells and a mouse keratinocytederived cell line, Hel37, as model systems for early development and skin, respectively. Like most connexin genes, Gjb3 is composed of two exons, the second of which contains the whole coding region and is separated from the first exon by an intron of 2.3 kb. Expression in keratinocytes is regulated by a basal promoter extending to 561 bp upstream of exon 1 in conjunction with a regulatory region between upstream positions 561 and 841. In contrast, expression of Gjb3 in embryonic stem cells depended on the basal promoter together with the intron. The enhancing effect of the ntron was found only in embryonic stem cells and depended on its native position and the integrity of the splice sites. Thus, expression of Gjb3 in keratinocytes and embryonic stem cells is regulated by different cis -regulatory elements and differs in its requirements for the intron in situ .

Several mutant mice have been generated to model connexin (Cx)-linked skin diseases; however, the role of connexins in skin maintenance and during wound healing remains to be fully elucidated. Here we generated a novel, viable, and fertile mouse (Cx26CK14-S17F/+) with the keratitis-ichthyosis-deafness mutant (Cx26S17F) driven by the cytokeratin 14 promoter. This mutant mouse mirrors several Cx26-linked human skin pathologies suggesting that the etiology of Cx26-linked skin disease indeed stems from epidermal expression of the Cx26 mutant. Cx26CK14-S17F/+ foot pad epidermis formed severe palmoplantar keratoderma, which expressed elevated levels of Cx26 and filaggrin. Primary keratinocytes isolated from Cx26CK14-S17F/+ neonates exhibited reduced gap junctional intercellular communication and migration. Furthermore, Cx26CK14-S17F/+ mouse skin wound closure was normal but repaired epidermis appeared hyperplastic with elevated expression of cytokeratin 6. Taken together, we suggest that the Cx26S17F mutant disturbs keratinocyte differentiation and epidermal remodeling following wound closure. We further posit that Cx26 contributes to epidermal homeostasis by regulating keratinocyte differentiation, and that mice harboring a disease-linked Cx26 mutant display epidermal abnormalities yet retain most wound healing properties.

Mutations in the human GJB3 gene that codes for Connexin31 (Cx31), a protein subunit of gap junction channels, have recently been reported to cause deafness and the skin disorder erythrokeratodermia variabilis. To study the function of this gene in mice, we generated animals with targeted replacement of the Cx31 gene (Gjb3) by a lacZ reporter gene. Although homozygous Cx31-deficient adult mice (Gjb3(-/-)) were found among the offspring of heterozygous Cx31-deficient parents (Gjb3(+/-)), 60% of the animals expected according to Mendelian inheritance were lost between ED 10.5 and 13.5. Placentas of Gjb3(-/-) embryos at ED 9.5 were smaller than controls as a result of severely reduced labyrinth and spongiotrophoblast size. From ED 10.5 onward, placentas of surviving Gjb3(-/-) embryos recovered progressively and reached normal size and morphology by ED 18.5. This corresponds to a time period in which another connexin isoform, Connexin43, is upregulated in spongiotrophoblast cells of Cx31-deficient and control placentas. No morphological or functional defects of skin or inner ear were observed in surviving adult Gjb3(-/-) mice. We conclude that Cx31 is essential for early placentation but can be compensated for by other connexins in the embryo proper and adult mouse.

To evaluate the influence of intracellular domains of connexin (Cx) on channel transfer properties, we analyzed mouse connexin (Cx) Cx26 and Cx30, which show the most similar amino acid sequence identities within the family of gap junction proteins. These connexin genes are tightly linked on mouse chromosome 14. Functional studies were performed on transfected HeLa cells stably expressing both mouse connexins. When we examined homotypic intercellular transfer of microinjected neurobiotin and Lucifer yellow, we found that gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer yellow. Furthermore, we observed heterotypic transfer of neurobiotin between Cx30-transfectants and HeLa cells expressing mouse Cx30.3, Cx40, Cx43 or Cx45, but not between Cx26 transfectants and HeLa cells of the latter group. The main differences in amino acid sequence between Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-terminal region of these integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30 chimeric cDNAs, which were also expressed in HeLa cells after transfection. Homotypic intercellular transfer of injected Lucifer yellow was observed exclusively with those chimeric constructs that coded for both cytoplasmic domains of Cx26 in the Cx30 backbone polypeptide chain. In contrast, cells transfected with a construct that coded for the Cx26 backbone with the Cx30 cytoplasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these connexins. In turn, the cytoplasmic loop and C-terminal domain of Cx30 prevent Lucifer yellow transfer when swapped with the corresponding domains of Cx26. In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal domains had been exchanged, the unitary channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly reduced. This suggests that these cytoplasmic domains interfere directly or indirectly with the diffusivity, the conductance and voltage gating of the channels.

Here we describe several gene targeting approaches currently used in our laboratory for the generation of deletion or replacement mutants of connexin genes in the mouse and discuss the advantage of the double-replacement strategy for the generation of conditional mutants. For the analysis of complementary functions of connexins, it will be necessary to generate mice with mutations in several connexin genes. We also report how this can be effectively accomplished. The replacement of targeted connexin-coding DNA with a reporter gene, to mimic expression of the deleted gene product, is currently being used in several laboratories. The use of different reporter genes or their differently localized gene products could allow distinction of promoter activity in double or triple connexin mutant mice.

The mouse connexin 36 (Cx36) gene was mapped on chromosome 2 and an identical transcriptional start site was determined in brain and retina on exon I. Rabbit polyclonal antibodies to the presumptive cytoplasmic loop of the Cx36 protein recognized in immunohistochemical analyses Cx36 expression in the retina, olfactory bulb, hippocampus, inferior olive and cerebellum. In olivary neurons strong punctate labeling at dendritic cell contacts and weaker labeling in the cytoplasm of dendrites were shown by immuno electron microscopy. After expression of mouse Cx36 cDNA in human HeLa cells, neurobiotin transfer was increased 1.8-fold and electrical conductance at least 15-fold compared to untransfected HeLa cells. No Lucifer Yellow transfer was detected in either untransfected or Cx36 transfected HeLa cells. Single Cx36 channels in transfected HeLa cells showed a unitary conductance of 14.3 + or - 0. 8 pS. The sensitivity of Cx36 channels to transjunctional voltage was low in both HeLa-Cx36 cells and Xenopus oocytes expressing mouse Cx36. No increased transfer of neurobiotin was detected in heterotypic gap junctions formed by Cx36 and 9 other connexins expressed in HeLa cells. Our results suggest that Cx36 channels function as electrical synapses for transmission of electrical and metabolic signals between neurons in the central nervous system.

Cardiac Conduction in C×40−/− Mice. Introduction: The gap junction protein connexin40 (C×40) normally is expressed in the murine atrial myocardium and ventricular conduction system. In mice lacking C×40, several changes in the surface ECG have been described. In this study, we analyzed cardiac conduction in more detail. Methods and Results: In open chest mice under urethane anesthesia, epicardial electrodes were used to determine a number of atrial and ventricular pacing parameters. The corrected sinus node recovery time was significantly longer in C×40−/− mice than in C×40+/+ mice (44.4 ± 7.2 msec vs 35.5 ± 8.0 msec). In addition, the Wenckebach period was longer in C×40−/− mice compared with the wild type (84.6 ± 5.4 msec vs 78.8 ± 3.6 msec), with the AV node probably limiting AV conduction in both cases. Whereas arrhythmias could not be induced by ventricular burst pacing in any of the mice, atrial burst pacing induced atrial tachyarrhythmias in 5 of 10 C×40−/− mice, but not in any of 9 Cx40−/− mice. Conduction velocities were measured in vivo using an array of unipolar recording electrodes. Ventricular conduction velocity did not differ between the groups, but atrial conduction velocity was reduced by 30% in C×40−/− mice compared with the wild type. Heterozygous C×40+/− mice did not differ significantly from the wild type in any respect. Conclusion: These findings indicate that in the atria and the AV conduction system, C×40 is an important determinant of conduction.

A new mouse connexin gene has been isolated that codes for a connexin protein of 505 amino acid residues. Based on the predicted molecular mass of 57.115 kDa, it has been designated connexin-57. Similar to most other mouse connexin genes, the coding region of connexin-57 is not interrupted by introns and exists in the mouse genome as a single-copy gene. Within the connexin family, this new gene shows highest sequence identity to porcine connexin-60 in the alpha group of connexins. The connexin-57 gene was mapped to a position on mouse chromosome 4, 30 centimorgans proximal to a cluster of previously mapped connexin genes. Low levels of connexin-57 mRNA were detected in skin, heart, kidney, testis, ovary, intestine, and in the mouse embryo after 8 days post coitum, but expression was not detected in brain, sciatic nerve or liver. In order to analyze gene function, the connexin-57 coding region was expressed by transfection in human HeLa cells, where it restored homotypic intercellular transfer of microinjected neurobiotin. Heterotypic transfer was observed between HeLa connexin-57 transfectants and HeLa cells, expressing murine connexin-43, -37, or -30.3. Double whole-cell voltage clamp analyses revealed that HeLa-connexin-57 transfectants expressed about 10 times more channels than parental HeLa cells. Voltage gating by transjunctional and transmembrane voltages as well as unitary conductance ( approximately 27 picosiemens) were different from intrinsic connexin channels in parental HeLa cells.

In rat brain, expression of the gap junction protein connexin30 increased during the first 3 weeks after birth and reached its maximum after 4 weeks, as shown by analysis with specific connexin30 antibodies. This contrasts with the prenatal onset of connexin43 expression. On cryosections of rat brain, connexin30 immunoreactivity was found near blood vessels and in ependymal as well as in leptomeningeal cells. Expression in the neuropil was first noticed 3 weeks after birth, showing the same spatial pattern of immunoreactivity as connexin43. This late onset of connexin30 expression in astrocytes was also seen in long-term glial cell cultures, where connexin30 was coexpressed with the astrocytic marker proteins S-100beta and glial fibrillary acid protein. In acute brain slices, connexin30 immunofluorescent signals were detected on processes of functionally identified astrocytes. Thus, our results show that connexin30 is expressed in three different cell types of the rodent brain. The late onset of connexin30 expression in astrocytes suggests that this gap junctional protein fulfills a role in intercellular communication among mature astrocytes.

The connexins are a family of proteins that form the intercellular membrane channels of gap junctions. Genes encoding 13 different rodent connexins have been cloned and characterized to date. Connexins vary both in their distribution among adult cell types and in the properties of the channels that they form. In order to explore the functional significance of connexin diversity, several mouse connexin-encoding genes have been disrupted by homologous recombination in embryonic stem cells. Although those experiments have illuminated specific physiological roles for individual connexins, the results have also raised the possibility that connexins may functionally compensate for one another in cells where they are coexpressed. In the present study, we have tested this hypothesis by interbreeding mice carrying null mutations in the genes (Gjb1 and Gja1) encoding connexin32 (β1 connexin) and connexin43 (α1 connexin), respectively. We found that fetuses lacking both connexins survive to term but, as expected, the pups die soon thereafter from the cardiac abnormality caused by the absence of connexin43. A survey of the major organ systems of the doubly mutant fetuses, including the thyroid gland, developing teeth, and limbs where these two connexins are coexpressed, failed to reveal any morphological abnormalities not already seen in connexin43 deficient fetuses. Furthermore, the production of thyroxine by doubly mutant thyroids was confirmed by immunocytochemistry. We conclude that, at least as far as the prenatal period is concerned, the normal development of those three organs in fetuses lacking connexin43 cannot simply be explained by the additional presence of connexin32 and vice-versa. Either gap junctional coupling is dispensable in embryonic and fetal cells in which these two connexins are coexpressed, or coupling is provided by yet another connexin when both are absent. Dev. Genet. 24:5–12, 1999. © 1999 Wiley-Liss, Inc.

Gap junctions connect neighboring cells via intercellular channels composed of connexins (Cx). Connexin 32 (Cx32) is the main connexin in hepatocytes. Gap junctions propagate a signal from periportal to perivenous hepatocytes generated by electrical stimulation of sympathetic liver nerves. Therefore, it was the aim of this study to examine the involvement of hepatocellular gap junctions in hormonal regulation. In perfused livers from wild-type mice and Cx32-deficient mice, the stimulation of glucose release by varying noradrenaline and glucagon concentrations was investigated. At saturating hormone concentrations, glucose release was the same in wild-type and Cx32-deficient livers. However, glucose output was significantly smaller in Cx32-deficient than wild-type livers at half-maximally effective hormone concentrations. Because the two hormones circulate at less than half-saturating concentrations and because they are degraded during passage of blood through the liver, they lose efficiency from the periportal to the perivenous zone. In wild-type livers, this decrease in efficiency can be partially compensated by intercellular signal propagation through gap junctions, resulting in higher hormone actions than in Cx32-deficient livers. It is concluded that gap junctions are not only involved in intercellular propagation of nervous, but also of hormonal signals from periportal to perivenous hepatocytes.