Identification of cells expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in gap junctions of rat brain and spinal cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with "permissive" connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

Activity-dependent neuronal control of gap-junctional communication in astrocytes

A typical feature of astrocytes is their high degree of intercellular communication through gap junction channels. Using different models of astrocyte cultures and astrocyte/neuron cocultures, we have demonstrated that neurons upregulate gap-junctional communication and the expression of connexin 43 (Cx43) in astrocytes. The propagation of intercellular calcium waves triggered in astrocytes by mechanical stimulation was also increased in cocultures. This facilitation depends on the age and number of neurons, indicating that the state of neuronal differentiation and neuron density constitute two crucial factors of this interaction. The effects of neurons on astrocytic communication and Cx43 expression were reversed completely after neurotoxic treatments. Moreover, the neuronal facilitation of glial coupling was suppressed, without change in Cx43 expression, after prolonged pharmacological treatments that prevented spontaneous synaptic activity. Altogether, these results demonstrate that neurons exert multiple and differential controls on astrocytic gap-junctional communication. Since astrocytes have been shown to facilitate synaptic efficacy, our findings suggest that neuronal and astrocytic networks interact actively through mutual setting of their respective modes of communication.     

Identification of Cells Expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in Gap Junctions of Rat Brain and Spinal Cord

We have identified cells expressing Cx26, Cx30, Cx32, Cx36 and Cx43 in gap junctions of rat central nervous system (CNS) using confocal light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling (FRIL). Confocal microscopy was used to assess general distributions of connexins, whereas the 100-fold higher resolution of FRIL allowed co-localization of several different connexins within individual ultrastructurally-defined gap junction plaques in ultrastructurally and immunologically identified cell types. In >4000 labeled gap junctions found in >370 FRIL replicas of gray matter in adult rats, Cx26, Cx30 and Cx43 were found only in astrocyte gap junctions; Cx32 was only in oligodendrocytes, and Cx36 was only in neurons. Moreover, Cx26, Cx30 and Cx43 were co-localized in most astrocyte gap junctions. Oligodendrocytes shared intercellular gap junctions only with astrocytes, and these heterologous junctions had Cx32 on the oligodendrocyte side and Cx26, Cx30 and Cx43 on the astrocyte side. In 4 and 18 day postnatal rat spinal cord, neuronal gap junctions contained Cx36, whereas Cx26 was present in leptomenigeal gap junctions. Thus, in adult rat CNS, neurons and glia express different connexins, with “permissive” connexin pairing combinations apparently defining separate pathways for neuronal vs. glial gap junctional communication.     

A Neuroprotective Role for Gap Junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

A neuroprotective role for gap junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

Connexin 30 Expression and Frequency of Connexin Heterogeneity in Astrocyte Gap Junction Plaques Increase with Age in the Rat Retina

We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05) but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05). With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.     

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha- type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.     

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.     

Connexin phosphorylation as a regulatory event linked to gap junction channel assembly

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells and are important in development and maintenance of cell homeostasis. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the cell cycle and the connexin “lifecycle”, such as trafficking, assembly/disassembly, degradation, as well as in the gating of “hemi” channels or intact gap junction channels. This review focuses on how phosphorylation can regulate the early stages of the connexin life cycle through assembly of functional gap junctional channels. The availability of sequences from the human genome databases has indicated that the number of connexins in the gene family is approximately 20, but we know mostly about how connexin43 (Cx43) is regulated. Recent technologies and investigations of interacting proteins have shown that activation of several kinases including protein kinase A, protein kinase C (PKC), p34^cdc2/cyclin B kinase, casein kinase 1 (CK1), mitogen-activated protein kinase (MAPK) and pp60^src kinase can lead to phosphorylation of the majority of the 21 serine and two of the tyrosine residues in the C-terminal region of Cx43. While many studies have correlated changes in kinase activity with changes in gap junctional communication, further research is needed to directly link specific phosphorylation events with changes in connexin oligomerization and gap junction assembly.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

SRC utilizes Cas to block gap junctional communication mediated by connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.     

Increased interaction of connexin43 with zonula occludens-1 during inhibition of gap junctions by G protein-coupled receptor agonists

Astrocytes are extensively coupled through gap junctions (GJs) that are composed of channels mostly constituted by connexin43 (Cx43). This astroglial gap junctional intercellular communication (GJIC) allows propagation of ions and signaling molecules critical for neuronal activity and survival. It is drastically inhibited by a short-term exposure to endothelin-1 (ET-1) or to sphingosine-1-phosphate (S1P), both compounds being inflammatory mediators acting through activation of GTP-binding protein-coupled receptors (GPCRs). Previously, we have identified the GTPases G(i/o) and Rho as key actors in the process of S1P-induced inhibition. Here, we asked whether similar mechanisms underlied the effects of ET-1 and S1P by investigating changes in the phosphorylation status of Cx43 and in the molecular associations of Cx43 with zonula occludens (ZO) proteins and occludin. We showed that the inhibitory effect of ET-1 on GJIC was entirely dependent on the activation of G(i/o) but not on Rho and Rho-associated kinase. Both ET-1 and S1P induced dephosphorylation of Cx43 located at GJs through a process mediated by G(i/o) and calcineurin. Thanks to co-immunoprecipitation approaches, we found that a population of Cx43 (likely junctional Cx43) was associated to ZO-1-ZO-2-occludin multiprotein complexes and that acute treatments of astrocytes with ET-1 or S1P induced a G(i/o)-dependent increase in the amount of Cx43 linked to these complexes. As a whole, this study identifies a new mechanism of GJIC regulation in which two GPCR agonists dynamically alter interactions of Cx43 with its molecular partners.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Gap junctions between cells expressing connexin 43 or 32 show inverse permselectivity to adenosine and ATP

Gap junctions, composed of proteins from the connexin family, are the only channels that directly connect the cytoplasm of adjacent cells to allow for the intercellular transfer of small hydrophilic molecules. Gap junctional communication is essential for proper development and health in animals and humans. Whereas the study of biological molecules that pass through gap junctions is extremely important, the identification of endogenous transjunctional metabolites is challenging. To help address this problem, we have developed a layered culture system to identify and quantitate the transfer of endogenous molecules that pass between cells through gap junctions. Using these techniques, we have identified several endogenous molecules that showed differential transfer between channels composed of Cx32 versus Cx43. For example, adenosine passed about 12-fold better through channels formed by Cx32. In contrast, AMP and ADP passed about 8-fold better, and ATP greater than 300-fold better, through channels formed by Cx43. Thus, addition of phosphate to adenosine appears to shift its relative permeability from channels formed by Cx32 to channels formed by Cx43. This suggests functional consequence because the energy status of a cell could be controlled via connexin expression and channel formation.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Gap junction assembly: multiple connexin fluorophores identify complex trafficking pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells

Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions.     

Connexin expression and gap junctional coupling in human cumulus cells: contribution to embryo quality

Gap junctional coupling among cumulus cells is important for oogenesis since its deficiency in mice leads to impaired folliculogenesis. Multiple connexins (Cx), the subunits of gap junction channels, have been found within ovarian follicles in several species but little is known about the connexins in human follicles. The aim of this study was to determine which connexins contribute to gap junctions in human cumulus cells and to explore the possible relationship between connexin expression and pregnancy outcome from in vitro fertilization (IVF). Cumulus cells were obtained from IVF patients undergoing intra-cytoplasmic sperm injection (ICSI). Connexin expression was examined by RT-PCR and confocal microscopy. Cx43 was quantified by immunoblotting and gap junctional coupling was measured by patch-clamp electrophysiology. All but 5 of 20 connexin mRNAs were detected. Of the connexin proteins detected, Cx43 forms numerous gap junction-like plaques but Cx26, Cx30, Cx30.3, Cx32 and Cx40 appeared to be restricted to the cytoplasm. The strength of gap junctional conductance varied between patients and was significantly and positively correlated with Cx43 level, but neither was correlated with patient age. Interestingly, Cx43 level and intercellular conductance were positively correlated with embryo quality as judged by cleavage rate and morphology, and were significantly higher in patients who became pregnant than in those who did not. Thus, despite the presence of multiple connexins, Cx43 is a major contributor to gap junctions in human cumulus cells and its expression level may influence pregnancy outcome after ICSI.     

Connexin-43 Interactions with ZO-1 and α- and β-tubulin

Gap junctions are composed of connexins that form transmembrane channels between adjacent cells. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating. Interestingly, channel-independent processes regulated by Cx43 have also been postulated. In our studies to elucidate the mechanism of Cx43 channel gating by growth factors and to explore additional functions of gap junctions, we have identified three interacting partners of the C-terminal tail of Cx43 (Cx43CT). (i) the c-Src tyrosine kinase, which phosphorylates Cx43CT and is involved in G protein-mediated inhibition of Cx43 gap junctional communication, (ii) the ZO-1 ‘scaffold’ protein, which might recruit signaling proteins into Cx43-based gap junctions. (iii) microtubules (consisting of α/β-tubulin dimers), which extend with their distal ends to Cx43-based gap junctions, suggesting that Cx43 gap junctions may play a novel role in regulating microtubule stability in contacted cells. Here we show that Cx43 binds α-tubulin equally well as β-tubulin. In addition, we show that the second, but not the first, PDZ domain of ZO-1 binds directly to Cx43, and we confirm that the very C-terminal isoleucine residue of Cx43 is critical for ZO-1 binding.     

Hyperglycaemia and diabetes impair gap junctional communication among astrocytes

Sensory and cognitive impairments have been documented in diabetic humans and animals, but the pathophysiology of diabetes in the central nervous system is poorly understood. Because a high glucose level disrupts gap junctional communication in various cell types and astrocytes are extensively coupled by gap junctions to form large syncytia, the influence of experimental diabetes on gap junction channel-mediated dye transfer was assessed in astrocytes in tissue culture and in brain slices from diabetic rats. Astrocytes grown in 15–25 mmol/l glucose had a slow-onset, poorly reversible decrement in gap junctional communication compared with those grown in 5.5 mmol/l glucose. Astrocytes in brain slices from adult STZ (streptozotocin)-treated rats at 20–24 weeks after the onset of diabetes also exhibited reduced dye transfer. In cultured astrocytes grown in high glucose, increased oxidative stress preceded the decrement in dye transfer by several days, and gap junctional impairment was prevented, but not rescued, after its manifestation by compounds that can block or reduce oxidative stress. In sharp contrast with these findings, chaperone molecules known to facilitate protein folding could prevent and rescue gap junctional impairment, even in the presence of elevated glucose level and oxidative stress. Immunostaining of Cx (connexin) 43 and 30, but not Cx26, was altered by growth in high glucose. Disruption of astrocytic trafficking of metabolites and signalling molecules may alter interactions among astrocytes, neurons and endothelial cells and contribute to changes in brain function in diabetes. Involvement of the microvasculature may contribute to diabetic complications in the brain, the cardiovascular system and other organs.