Connexin Family Of Proteins Research Articles

Connexinplexity: the spatial and temporal expression of connexin genes during vertebrate organogenesis.

Animal development requires coordinated communication between cells. The Connexin family of proteins is a major contributor to intercellular communication in vertebrates by forming gap junction channels that facilitate the movement of ions, small molecules, and metabolites between cells. Additionally, individual hemichannels can provide a conduit to the extracellular space for paracrine and autocrine signaling. Connexin-mediated communication is widely used in epithelial, neural, and vascular development and homeostasis, and most tissues likely use this form of communication. In fact, Connexin disruptions are of major clinical significance contributing to disorders developing from all major germ layers. Despite the fact that Connexins serve as an essential mode of cellular communication, the temporal and cell-type-specific expression patterns of connexin genes remain unknown in vertebrates. A major challenge is the large and complex connexin gene family. To overcome this barrier, we determined the expression of all connexins in zebrafish using single-cell RNA-sequencing of entire animals across several stages of organogenesis. Our analysis of expression patterns has revealed that few connexins are broadly expressed, but rather, most are expressed in tissue- or cell-type-specific patterns. Additionally, most tissues possess a unique combinatorial signature of connexin expression with dynamic temporal changes across the organism, tissue, and cell. Our analysis has identified new patterns for well-known connexins and assigned spatial and temporal expression to genes with no-existing information. We provide a field guide relating zebrafish and human connexin genes as a critical step toward understanding how Connexins contribute to cellular communication and development throughout vertebrate organogenesis.

The Role of Connexin Hemichannels in Inflammatory Diseases.

Simple SummaryConnexin hemichannels are formed by connexin protein family members, and connect cytosol and extracellular milium. The function of connexin hemichannels cannot be readily distinguished from the gap junctions formed also by connexins, or hemichannels formed by pannexins. It appears that connexin hemichannels are normally closed to maintain cellular homeostasis, but can be activated in pathophysiological processes to serve as toxic membrane pores. On the other hand, gap junctions are normally open in order to perform critical physiological functions, but are often closed or down-regulated under pathological conditions. The development and characterization of connexin mimetic peptides have resulted in a panel of connexin hemichannel-selective blockers. Investigations using these blockers have shown that the opening of connexin hemichannels facilitates the release of damage-associated molecular patterns, a class of endogenous molecules that are critical for the pathogenesis of inflammatory diseases. The blockade of connexin hemichannels virtually always leads to attenuated inflammation, reduced tissue injury and improved organ function. In this review, we provide an updated view of the role of connexin hemichannels in inflammatory diseases.The connexin protein family consists of approximately 20 members, and is well recognized as the structural unit of the gap junction channels that perforate the plasma membranes of coupled cells and, thereby, mediate intercellular communication. Gap junctions are assembled by two preexisting hemichannels on the membranes of apposing cells. Non-junctional connexin hemichannels (CxHC) provide a conduit between the cell interior and the extracellular milieu, and are believed to be in a protectively closed state under physiological conditions. The development and characterization of the peptide mimetics of the amino acid sequences of connexins have resulted in the development of a panel of blockers with a higher selectivity for CxHC, which have become important tools for defining the role of CxHC in various biological processes. It is increasingly clear that CxHC can be induced to open by pathogen-associated molecular patterns. The opening of CxHC facilitates the release of damage-associated molecular patterns, a class of endogenous molecules that are critical for the pathogenesis of inflammatory diseases. The blockade of CxHC leads to attenuated inflammation, reduced tissue injury and improved organ function in human and animal models of about thirty inflammatory diseases and disorders. These findings demonstrate that CxHC may contribute to the intensification of inflammation, and serve as a common target in the treatments of various inflammatory diseases. In this review, we provide an update on the progress in the understanding of CxHC, with a focus on the role of these channels in inflammatory diseases.

Expression of “Connexin 43” in Colorectal Carcinomas: Histopathological and Immunohistochemical Study

BACKGROUND: Colorectal cancer is one of the most common cancers worldwide and leading cause of cancer related deaths. Connexins are integral membrane proteins that form channels between adjacent cells. Gap junction intercellular communication plays essential roles in tissue homoeostasis and regulation of cell growth and differentiation. Connexins can act as either tumor suppressors or tumor promoters. The human connexin protein family contains 21 members, of which the most widely studied is connexin 43 (Cx 43).
 OBJECTIVES: Investigation of immunohistochemical expression of Cx 43 in cases of colorectal adenoma and carcinoma and correlation of this expression with the clinico-pathological aspects of the tumors.
 MATERIALS AND METHODS: Seventy formalin fixed paraffin embedded BC tissue sections were randomly collected. All the available data were collected from the patients’ reports. The paraffin blocks were sectioned and stained with hematoxylin and eosin stains for histologic evaluation. Additional sections were immunostained with Cx 43.
 RESULTS: Cx 43 expression was negative in all studied cases.
 CONCLUSION: Cx 43 is a tumor suppressor that is lost early in colorectal carcinogenesis and can be considered as potential target for cancer chemoprevention and chemotherapy aiming at restoration of normal connexin expression and functional gap junctions.

Connexin 43 reduces susceptibility to sympathetic atrial fibrillation.

Atrial fibrillation (AF) is the most common arrhythmia reported in clinical practice. Connexin 43 (Cx43) is a member of the connexin protein family, which serves important roles in signal transduction in vivo. The aim of the present study was to investigate the role of Cx43 in the induction and maintenance of atrial fibrillation by using an animal model of sympathomimetic atrial fibrillation. Cx43 was successfully knocked down in the myocardium with gene‑specific small interfering (si)RNA via lentiviral infection. A total of 25dogs were randomly and evenly divided into five groups: Normal(N), rapid atrial pacing (RAP), isoproterenol (ISO)+RAP, RAP +Cx43 siRNA and ISO +RAP+Cx43 siRNA. The mRNA and protein levels, as well as the distribution of Cx43 on the cell membrane, were gradually decreased in each group compared with the N group following treatment (P<0.05). The induction rate of the atrial effective refractory period was not significantly affected in the RAP and RAP+Cx43 siRNA groups, whereas it was significantly reduced in the ISO+RAP and ISO+RAP+Cx43 siRNA groups compared with the N group (P<0.05). The induction rate of AF was gradually increased in the RAP+Cx43 siRNA, ISO+RAP and ISO+RAP+Cx43 siRNA groups compared with the Ngroup (P<0.05). The expression of nerve growth factor (NGF) and tyrosine hydroxylase (TH) was gradually increased in the ISO+RAP and ISO+RAP+Cx43 siRNA groups compared with their respective controls (RAP and RAP+Cx43 siRNA groups, respectively). However, no significant difference in the levels of NGF and TH was observed between the RAP, RAP + Cx43 siRNA, ISO + RAP and ISO + RAP + Cx43 siRNA groups. The mitochondrial morphology in each group was notably altered compared with the N group. The mitochondrial reactive oxygen species production and apoptotic index were gradually increased in each group compared with the N group (P<0.05). The results of the present study suggest that Cx43 reduces susceptibility to AF. Downregulation of Cx43 mediates the induction and maintenance of sympathetic AF.

Spatiotemporal expression pattern of Connexin 43 during early chick embryogenesis

During embryogenesis, a single cell develops into new tissues and organs that are made up of a number of different cell types. The assembly of the trigeminal ganglion (cranial nerve V), an important component of the peripheral nervous system, typifies this process. The trigeminal ganglia perform key sensory functions, including sensing pain and touch in the face, and arise from cells of two different progenitor populations, the neural crest and the cranial placodes. One question that remains poorly understood is how these two populations of cells interact with each other during development to form a functional ganglion. Gap junctions are intercellular channels that allow for the passage of small solutes between connected cells and could serve as one potential mechanism by which neural crest and placode cells communicate to create the trigeminal ganglia. To this end, we have generated a comprehensive spatiotemporal expression profile for the gap junction protein Connexin 43, a highly expressed member of the Connexin protein family during development. Our results reveal that Connexin 43 is expressed in the neural folds during neural fold fusion and in premigratory neural crest cells prior to the epithelial-to-mesenchymal transition (EMT), during EMT, and in migratory neural crest cells. During trigeminal gangliogenesis, Connexin 43 is expressed in cranial neural crest cells and the mesenchyme but is strikingly absent in the placode-derived neurons. These data underscore the complexity of bringing two distinct cell populations together to form a new tissue during development and suggest that Connexin 43 may play a key role within neural crest cells during EMT, migration, and trigeminal gangliogenesis.

Physiological roles of connexins in labour and lactation.

The connexin family of proteins are best known as oligomerizing to form intercellular membrane channels (gap junctions) that metabolically and ionically couple cells to allow for coordinated cellular function. Nowhere in the body is this role better illustrated than in the uterine smooth muscle during parturition, where gap junctions conduct the contraction wave throughout the tissue to deliver the baby. Parturition is followed by the onset of lactation with connexins contributing to both the dramatic reorganization of mammary gland tissue leading up to lactation and the smooth muscle contraction of the myoepithelial cells which extrudes the milk. This review summarizes what is known about the expression and roles of individual connexin family members in the uterus during labour and in the mammary glands during development and lactation. Connexin loss or malfunction in mammary glands and the uterus can have serious implications for the health of both the mother and the newborn baby.

Electron Microscopic Analysis of Lens Gap Junction Connexin50 Hemichannels and Channels

The mammalian lens is an avascular organelle that depends on gap junction (GJ) mediated intercellular communication for homeostasis, growth and development. Connexin50 (Cx50) is one of three lens GJ proteins and one of the largest members of the connexin protein family. Cx50 is present in all cells of the lens, first synthesized in the epithelium and persisting at high levels in differentiating fibers. Cases of human hereditary cataracts caused by &gt;20 Cx50 distinct mutations demonstrate its crucial role in lens function and Cx50 knockout mice develop cataracts. Little is known about Cx50 structure except through homology with Cx26, a small connexin solved to 3.5 Å resolution by X‐ray crystallography and NMR of Cx50 C‐terminal peptides. Cx26 and Cx50 have strong sequence conservation and folding in their transmembrane and extracellular domains, but Cx26 lacks the extensive, non‐conserved C‐terminus present in Cx50. We have examined the full‐length Cx50 hemichannels and channels by single particle EM methods, the only structural biology technique that directly addresses how the large Cx50 C‐terminus is organized within the full‐length channel. Using baculovirus expression in Sf9 cells, we have purified both full‐length mouse Cx50 (440 amino acids) and a truncated form (Cx50del250‐440, 249 amino acids long) missing most of the C‐terminus such that it is slightly longer than Cx26 (226 amino acids). Electron micrographs of purified Cx50 hemichannels show a range of morphologies due to the way the oligomers are oriented on the grid. In contrast, Cx26 and Cx50del250‐440 channels are more homogeneous, displaying the canonical “doughnut‐like” appearance in projection because their structure.Funding: NIH grants GM065937 (GS) &amp; GM103412 (NCMIR)

Connexins in colorectal cancer pathogenesis.

The connexins constitute a family of integral membrane proteins that form channels between adjacent cells. These channels are assembled in plasma membrane domains known as gap junctions and enable cells to directly exchange ions and small molecules. Intercellular communication via gap junctions plays important roles in regulating cell growth and differentiation and in maintaining tissue homeostasis. This type of cell communication is often impaired during cancer development, and several members of the connexin protein family have been shown to act as tumor suppressors. Emerging evidence suggests that the connexin protein family has important roles in colorectal cancer development. In the normal colonic epithelial tissue, three connexin isoforms, connexin 26 (Cx26), Cx32 and Cx43, have been shown to be expressed at the protein level. Colorectal cancer development is associated with loss of connexin expression or relocalization of connexins from the plasma membrane to intracellular compartments. Downregulation of connexins in colorectal carcinomas at the transcriptional level involves cancer-specific promoter hypermethylation. Recent studies suggest that Cx43 may constrain growth of colon cancer cells by interfering with the Wnt/β-catenin pathway. There is also increasing evidence that the connexins may have potential as prognostic markers in colorectal cancer. This review discusses the role of connexins in colorectal cancer pathogenesis, as well as their potential as prognostic markers and targets in the prevention and treatment of the disease.

Requirement of neuronal connexin36 in pathways mediating presynaptic inhibition of primary afferents in functionally mature mouse spinal cord

Electrical synapses formed by gap junctions containing connexin36 (Cx36) promote synchronous activity of interneurones in many regions of mammalian brain; however, there is limited information on the role of electrical synapses in spinal neuronal networks. Here we show that Cx36 is widely distributed in the spinal cord and is involved in mechanisms that govern presynaptic inhibition of primary afferent terminals. Electrophysiological recordings were made in spinal cord preparations from 8- to 11-day-old wild-type and Cx36 knockout mice. Several features associated with presynaptic inhibition evoked by conditioning stimulation of low threshold hindlimb afferents were substantially compromised in Cx36 knockout mice. Dorsal root potentials (DRPs) evoked by low intensity stimulation of sensory afferents were reduced in amplitude by 79% and in duration by 67% in Cx36 knockouts. DRPs were similarly affected in wild-types by bath application of gap junction blockers. Consistent with presynaptic inhibition of group Ia muscle spindle afferent terminals on motoneurones described in adult cats, conditioning stimulation of an adjacent dorsal root evoked a long duration inhibition of monosynaptic reflexes recorded from the ventral root in wild-type mice, and this inhibition was antagonized by bicuculline. The same conditioning stimulation failed to inhibit monosynaptic reflexes in Cx36 knockout mice. Immunofluorescence labelling for Cx36 was found throughout the dorsal and ventral horns of the spinal cord of juvenile mice and persisted in mature animals. In deep dorsal horn laminae, where interneurones involved in presynaptic inhibition of large diameter muscle afferents are located, cells were extensively dye-coupled following intracellular neurobiotin injection. Coupled cells displayed Cx36-positive puncta along their processes. Our results indicate that gap junctions formed by Cx36 in spinal cord are required for maintenance of presynaptic inhibition, including the regulation of transmission from Ia muscle spindle afferents. In addition to a role in presynaptic inhibition in juvenile animals, the persistence of Cx36 expression among spinal neuronal populations in the adult mouse suggests that the contribution of electrical synapses to integrative processes in fully mature spinal cord may be as diverse as that found in other areas of the CNS.

DNA methylation analyses of the connexin gene family reveal silencing of GJC1 (Connexin45) by promoter hypermethylation in colorectal cancer

Gap junctions are specialized plasma membrane domains consisting of channels formed by members of the connexin protein family. Gap junctional intercellular communication is often lost in cancers due to aberrant localization or downregulation of connexins, and connexins are therefore suggested to act as tumor suppressor genes in various tissues. The aim of this study was to investigate the expression pattern and DNA promoter methylation status of connexins in colorectal cancer. Expression of six (GJA1, GJA9, GJB1, GJB2, GJC1 and GJD3) connexin genes was detected in normal colonic tissue samples. GJC1 expression was reduced in colorectal carcinomas compared to normal tissue samples. All analyzed connexins were hypermethylated in colon cancer cell lines, although at various frequencies. GJA4, GJB6 and GJD2 were hypermethylated in 60% (29/48), 25% (12/48) and 96% (46/48) of primary colorectal carcinomas, respectively. However, the methylation status was not associated with gene expression. GJC1 has two alternative promoters, which were methylated in 42% (32/76) and 38% (25/65) of colorectal tumors, and in none of the normal mucosa samples. Expression of GJC1 was significantly lower in methylated compared with unmethylated samples (p < 0.01) and was restored in cell lines treated with the demethylating drug 5-aza-2'deoxycytidine. Taken together, DNA hypermethylation of the promoter region of GJC1, encoding connexin45, is an important mechanism in silencing gene expression in colorectal cancer.

PI3K/Akt signaling is involved in the disruption of gap junctional communication caused by v-Src and TNF-α

Gap junctional communication, which is mediated by the connexin protein family, is essential for the maintenance of normal tissue function and homeostasis. Loss of intercellular communication results in a failure to coordinately regulate cellular functions, and it can facilitate tumorigenesis. Expression of oncogenes and stimulation with cytokines has been shown to suppress intercellular communication; however, the exact mechanism by which intercellular communication is disrupted by these factors remains uncertain. In this report, we show that Akt is essential for the disruption of gap junctional communication in v-Src-transformed cells. In addition, inhibition of Akt restores gap junctional communication after it is suppressed by TNF-α signaling. Furthermore, we demonstrate that the expression of a constitutively active form of Akt1, but not of Akt2 or Akt3, is sufficient to suppress gap junctional communication. Our results clearly define Akt1 as one of the critical regulators of gap junctional communication.

Calcium‐induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43

Carbenoxolone (Cbx), a substance from the medicinal licorice is used for anti‐inflammatory treatments. Cbx was already shown to induce opening of the permeability transition pore (PTP) in rat liver mitochondria (RLM). We investigated the mechanism of action of Cbx on Ca2+‐induced PTP opening in rat brain mitochondria (RBM), and in rat heart mitochondria (RHM) and in RLM, in an attempt to identify the molecular target of Cbx in mitochondria. In RBM, RHM and RLM, threshold Ca2+ load induced PTP opening. This was manifested by sudden Ca2+efflux from the mitochondrial matrix, a drop in membrane potential, and swelling of mitochondria. At 1 μM, Cbx enhanced the Ca2+‐induced, Cyclosporine A‐sensitive PTP opening. A well‐known target of Cbx in cells is the connexin family of proteins, which form gap junctions. Moreover, Connexin43 (Cx43) was previously found in RHM and attributed to the protective mechanism induced by ischemic preconditioning. We hypothesized that Cx43 might be a target for Cbx in brain mitochondria. We clearly detected Cx43 by Western blot in RBM and in mitochondria isolated from astrocytes, less in RHM, but not in RLM. Interestingly, the anti‐Cx43 antibody abolished the Cbx‐enhanced PTP opening in RBM, but not in RLM. In RBM, PTP caused serine‐368 phosphorylation of Cx43. These results show that Cbx facilitates Ca2+‐induced PTP opening, addressing as possible molecular target Cx43 in brain mitochondria.

Silver nanoparticles up-regulate Connexin43 expression and increase gap junctional intercellular communication in human lung adenocarcinoma cell line A549

Silver nanoparticles (Ag NPs) are increasingly being used in wound dressings, medical settings, and various household products due to their unique properties and antimicrobial activity. Despite the widespread use of Ag NP products, the molecular mechanisms underlying the biological effects of Ag NPs remain unclear. Gap junctional intercellular communication (GJIC), formed by the connexin protein family, plays a critical role in the maintenance of tissue and organ homeostasis. This study was undertaken to investigate the effects of well characterized, PVP-coated Ag NPs (69 ± 3 nm) and silver nitrate on GJIC and connexin43 (Cx43) expression in human lung adenocarcinoma cell line A549. Our results showed that Ag NPs increased GJIC in A549 cells as assayed by dye transfer method. Western blot analysis showed that incubation of cells with Ag NPs significantly increased the expression of Cx43 protein. In addition, Ag NPs up-regulated expression of Cx43 mRNA in a dose-dependent manner. Silver nitrate failed to increase GJIC and the expression of Cx43 protein. It, however, increased Cx43 mRNA expression in A549 cells. Taken together, our results provide the first evidence that Ag NPs induced the increase of GJIC activity in A549 cells through up-regulation of Cx43 protein, suggesting that Cx43 and GJIC may be one of the targets for Ag NPs biological effects.

Connexins as Precocious Markers and Molecular Targets for Chemical and Pharmacological Agents in Carcinogenesis

Gap junctions, intercellular channels structured by the connexin protein family, have been implicated in the control of cell homeostasis, proliferation, differentiation and death. A loss of the gap junction intercellular communication and/or connexin dysfunction are typical features of cancer per se and have been associated with the effect of many carcinogens. Indeed, many early human neoplasia of various organs and human tumor cell lines exhibit deficient connexin-mediated communication expression mainly related, in a large number of observations, with an aberrant cytoplasmic localization of this membranous protein. Restoration of normal phenotype in transformed cells by restoration of exogenous connexin gave rise to the concept that connexins may act as tumor suppressors. However, the mechanisms by which connexins mediate such a tumor suppressor effect are multiple. They may result from: formation of functional channels; hemichannels or are directly associated with connexin expression. In addition, the literature shows that they may be dependent upon the cell type and the connexin type. In the present review, we analyze all these aspects of connexin/gap junction involvement in the carcinogenesis process, in human cancers and discuss the possibility of using connexins as potential anti-oncogenic targets for cancer chemoprevention and/or chemotherapy.

Coincidence of mutations in different connexin genes in Hungarian patients

Mutations in the GJB2 gene are the most common cause of hereditary prelingual sensorineural hearing impairment in Europe. Several studies indicate that different members of the connexin protein family interact to form gap junctions in the inner ear. Mutations in different connexin genes may accumulate and, consequently lead to hearing impairment. Therefore, we screened 47 Hungarian GJB2- heterozygous (one mutation in coding exon of the GJB2 gene) patients with hearing impairment for DNA changes in two further connexin genes (GJB6 and GJB3) and in the 5' non-coding region of GJB2 including the splice sites. Eleven out of 47 GJB2-heterozygous patients analyzed carried the splice site mutation -3170G>A in the 5'UTR region of GJB2. One out of these 11 patients showed homozygous -3170G>A genotype in combination with p.R127H. Next to the GJB2 mutations we noted 2 cases of deletion in GJB6 [Delta(GJB6-D13S1830)] and 3 (2 new and 1 described) base substitutions in GJB3 [c.357C>T, c.798C>T and c.94C>T (p.R32W)] which are unlikely disease-causing. Our results suggest the importance of routine screening for the rather frequent -3170G>A mutation (in addition to c.35delG) in patients with hearing impairment.

Mutation of a Conserved Threonine in the Third Transmembrane Helix of α- and β-Connexins Creates a Dominant-negative Closed Gap Junction Channel

Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic end of the third transmembrane helix. This threonine is strictly conserved among members of the alpha- and beta-connexin subgroups but not the gamma-subgroup. In HeLa cells, connexin43 and connexin26 mutants are synthesized, traffic to the plasma membrane, and make gap junctions with the same overall appearance as wild type. We have isolated connexin26T135A gap junctions both from HeLa cells and baculovirus-infected insect Sf9 cells. By using cryoelectron microscopy and correlation averaging, difference images revealed a small but significant size change within the pore region and a slight rearrangement of the subunits between mutant and wild-type connexons expressed in Sf9 cells. Purified, detergent-solubilized mutant connexons contain both hexameric and partially disassembled structures, although wild-type connexons are almost all hexameric, suggesting that the three-dimensional mutant connexon is unstable. Mammalian cells expressing gap junction plaques composed of either connexin43T154A or connexin26T135A showed an absence of dye coupling. When expressed in Xenopus oocytes, these mutants, as well as a cysteine substitution mutant of connexin50 (connexin50T157C), failed to produce electrical coupling in homotypic and heteromeric pairings with wild type in a dominant-negative effect. This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo.

Gap junctions and the connexin protein family.

Gap junctions (Gj) form conduits between adjacent cells that are composed of connexin (Cx) protein subunits and allow direct intercellular communication. To date, the connexin gene family comprises 20 members in the mouse and 21 members in the human genome, 19 of which can be grouped as sequence-orthologous pairs. The structure of connexin genes is relatively simple. An untranslated exon 1 is separated by an intron of different length from exon 2, containing the uninterrupted coding region and the 3'-untranslated region (3'-UTR). However, in some connexin genes, the untranslated regions and the reading frame are spliced. Among the known "cardiovascular" connexins, Cx37 and Cx40 were demonstrated to be functionally expressed in mouse and human endothelial cells and Cx40, Cx43 as well as Cx45 in cardiomyocytes of both species. Functional properties, like permeabilities, charge selectivity and unitary conductivity were investigated after directed expression of these connexins in cultured cell lines or paired Xenopus oocytes. Targeted deletion of their coding sequence in the mouse genome allowed study of the biological relevance of Cx37, Cx40, Cx43 and Cx45 with regard to cardiovascular morphology and function. After ablation of Cx37 or Cx40, mice were viable and could be used to study defects in the adult cardiovascular system but loss of Cx43 or Cx45 led to neonatal or embryonic lethality, respectively. Conditional and cell-type specific deletion of both connexins in the heart or blood vessels can help to overcome this obstacle. As yet only little is known about mutations in the human genes for Cx37, Cx40, Cx43 and Cx45. Thus, a profound comparison between human and mouse phenotypes is not yet possible.

Connexin26 in adult rodent central nervous system: demonstration at astrocytic gap junctions and colocalization with connexin30 and connexin43.

The connexin family of proteins (Cx) that form intercellular gap junctions in vertebrates is well represented in the mammalian central nervous system. Among these, Cx30 and Cx43 are present in gap junctions of astrocytes. Cx32 is expressed by oligodendrocytes and is present in heterologous gap junctions between oligodendrocytes and astrocytes as well as at autologous gap junctions between successive myelin layers. Cx36 mRNA has been identified in neurons, and Cx36 protein has been localized at ultrastructurally defined interneuronal gap junctions. Cx26 is also expressed in the CNS, primarily in the leptomeningeal linings, but is also reported in astrocytes and in neurons of developing brain and spinal cord. To establish further the regional, cellular, and subcellular localization of Cx26 in neural tissue, we investigated this connexin in adult mouse brain and in rat brain and spinal cord using biochemical and immunocytochemical methods. Northern blotting, western blotting, and immunofluorescence studies indicated widespread and heterogeneous Cx26 expression in numerous subcortical areas of both species. By confocal microscopy, Cx26 was colocalized with both Cx30 and Cx43 in leptomeninges as well as along blood vessels in cortical and subcortical structures. It was also localized at the surface of oligodendrocyte cell bodies, where it was coassociated with Cx32. Freeze-fracture replica immunogold labeling (FRIL) demonstrated Cx26 in most gap junctions between cells of the pia mater by postnatal day 4. By postnatal day 18 and thereafter, Cx26 was present at gap junctions between astrocytes and in the astrocyte side of most gap junctions between astrocytes and oligodendrocytes. In perinatal spinal cord and in five regions of adult brain and spinal cord examined by FRIL, no evidence was obtained for the presence of Cx26 in neuronal gap junctions. In addition to its established localization in leptomeningeal gap junctions, these results identify Cx26 as a third connexin (together with Cx30 and Cx43) within astrocytic gap junctions and suggest a further level of complexity to the heterotypic connexin channel combinations formed at these junctions.

Diversity and molecular anatomy of gap junctions.

In animal tissues, most cells are connected via intercellular cytoplasmic channels called gap junctions. Various electron microscopy techniques have made a crucial contribution to our understanding of the function and structure of gap junction channels. Tracer studies and freeze-fracture replica observations indicate that the connexon, the unit gap junction channel, is a pair of hemichannels apposed in the narrow intercellular gap between neighboring cell membranes. Recent advances in cellular biology have shown that connexon hemichannels are composed of hexamers of connexin proteins. Purification of the gap junction membrane and cDNA cloning analysis indicate the diversity of the connexin protein family, which contains more than 18 members, and their tissue- and cell type-specific distributions. Defects in some connexin genes may cause various hereditary diseases, such as X-linked Charcot-Marie-Tooth disease (Cx32), nonsyndromic autosomal deafness (Cx26), and cataract (Cx50). Analysis of gene knockout mice indicates that certain types of connexin play important roles in differentiation and development at crucial times in specific tissues and cell types.

Expression of gap junction protein connexin-43 in the human and baboon (Papio anubis) corpus luteum.

In the nonhuman primate and human corpora lutea, gap junctions have been identified by means of electron microscopy. Gap junctions are formed by connexons, which consist of a multigene family of tissue-specific connexins. In the ovarian follicle, the gap junction protein connexin-43 is present and hormonally regulated. However, there is little evidence indicating the type of connexin present in the corpus luteum. Therefore, the aim of this study was to demonstrate the presence of gap junctions by electron microscopy and the presence of connexin-43 and messenger ribonucleic acid (mRNA) for this protein. Using immunocytochemical procedures, we have shown the presence of connexin-43 in baboon and human midluteal phase corpora lutea and in the atretic corpora lutea of the baboon. The intensity of immunoreactivity was lower in atretic corpora lutea than in the midluteal phase corpora lutea. Western analysis indicates the presence of two bands at 43-45 kDa, and that the levels of connexin-43 protein are abundant in the midluteal phase. The two bands suggest the presence of the protein in a phosphorylated or a nonphosphorylated form. Ribonuclease protection assay suggests that the mRNA levels of connexin-43 remain constant throughout the luteal phase. mRNA for connexin-43 was not detectable in atretic corpora lutea. Thus, connexin-43 is one of the connexin family of proteins forming the connexon of gap junctions in the baboon and human corpus luteum. The expression of the protein may be hormonally regulated by locally produced factors, such as estradiol and progesterone. We suggest that gap junctional communication between the cells of the primate and human corpus luteum may be important in hormone synthesis and secretion and may be involved in the process of luteolysis through luteal cell apoptosis.