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.     

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.     

Heterocellular gap junctional communication between alveolar epithelial cells

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.     

Gap junction proteins in inhibitory neurons of the adult barrel neocortex

Recent studies indicate that electrical coupling among cortical neurons may persist throughout development; electrophysiological recordings made in cortical slices from young rats reveal that numerous GABAergic neurons are electrically coupled. To determine whether these in vitro findings reflect an inhibitory neural circuit that could be functionally relevant in vivo in adult rodents, we sought to identify whether inhibitory, parvalbumin-containing neurons of the mature cortex express gap junction proteins. Immunohistochemistry was used to examine the laminar distribution of the gap junction-forming proteins connexin 32 (Cx32), connexin 36 (Cx36) and connexin 43 (Cx43) in the somatosensory cortex of the adult mouse. Double labeling immunofluorescence identified Cx32, Cx36 and Cx43 in cortical neurons that were immunoreactive (-ir) for the neuronal markers neurofilament 145 kDa and neuronal nuclei (NeuN). Parvalbumin-ir neurons throughout the cortical laminae were labeled with Cx32-ir, Cx36-ir and Cx43-ir. Stereological methods were used to quantify the extent of parvalbumin colocalization with connexins. Analysis indicated that approximately 40% of parvalbumin-ir neurons were double labeled with either Cx32-ir or Cx43-ir, and approximately 50% of parvalbumin-ir neurons were double labeled with Cx36. These findings establish an anatomical substrate for widespread electrical coupling of neurons in somatosensory cortex and suggest that gap junctions among inhibitory interneurons may persist into adulthood, providing an important mechanism for neuronal communication.     

N-cadherin and Cx43alpha1 gap junctions modulates mouse neural crest cell motility via distinct pathways

Our previous studies showed an essential role for connexin 43 or alpha1 connexin (Cx43alpha1) gap junctions in the modulation of neural crest cell motility. Cx43alpha1 gap junctions and N-cadherin containing adherens junctions are expressed in migrating cardiac neural crest cells. Analysis of the N-cadherin knockout (KO) mouse model revealed that N-cadherin is essential for gap junction mediated dye coupling but not for expression of Cx43alpha1 gap junctions in neural crest cells. Time lapse videomicroscopy and motion analysis showed that the motility of N-cadherin KO neural crest cells were altered, but the motility changes differed compared to Cx43alpha1 KO neural crest cells. These observations suggest that the role of N-cadherin in cell motility is not simply mediated via the modulation of Cx43alpha1 mediated cell-cell communication. This was confirmed by a parallel analysis of wnt-1 deficient neural crest cells, which also showed a reduction in dye coupling, and yet no change in cell motility. Analysis of p120 catenin (p120ctn), an Amardillo family protein known to play a role in cell motility, showed that it is colocalized with N-cadherin and Cx43alpha1 in migrating neural crest cells. This subcellular distribution was altered in the N-cadherin and Cx43alpha1 KO neural crest cells. Given these results, we propose that N-cadherin and Cx43alpha1 may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.     

N-Cadherin and Cx43α1 Gap Junctions Modulates Mouse Neural Crest Cell Motility via Distinct Pathways

Our previous studies showed an essential role for connexin 43 or α₁ connexin (Cx43α1) gap junctions in the modulation of neural crest cell motility. Cx43α1 gap junctions and N-cadherin containing adherens junctions are expressed in migrating cardiac neural crest cells. Analysis of the N-cadherin knockout (KO) mouse model revealed that N-cadherin is essential for gap junction mediated dye coupling but not for expression of Cx43α1 gap junctions in neural crest cells. Time lapse videomicroscopy and motion analysis showed that the motility of N-cadherin KO neural crest cells were altered, but the motility changes differed compared to Cx43α1 KO neural crest cells. These observations suggest that the role of N-cadherin in cell motility is not simply mediated via the modulation of Cx43α1 mediated cell-cell communication. This was confirmed by a parallel analysis of wnt-1 deficient neural crest cells, which also showed a reduction in dye coupling, and yet no change in cell motility. Analysis of p 120 catenin (p 120ctn), an Amardillo family protein known to play a role in cell motility, showed that it is colocalized with N-cadherin and Cx43α1 in migrating neural crest cells. This subcellular distribution was altered in the N-cadherin and Cx43α1 KO neural crest cells. Given these results, we propose that N-cadherin and Cx43α1 may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.     

Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/- mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/- mice was due almost entirely to a reduction in the number of individual gap junctions (226 +/- 52 vs. 150 +/- 32 individual gap junctions/field in Cx43 +/+ and +/- ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.     

Heart and head defects in mice lacking pairs of connexins

Gene ablation studies in mice have revealed roles for gap junction proteins (connexins) in heart development. Of the 20 connexins in vertebrates, four are expressed in developing heart: connexin37 (Cx37), connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). Although each cardiac connexin has a different pattern of expression, some heart cells coexpress multiple connexins during cardiac morphogenesis. Since different connexins could have overlapping functions, some developmental phenotypes may only become evident when more than one connexin is ablated. In this study, we interbred Cx40(-/-) and Cx43(-/-) mice to generate mice lacking both Cx40 and Cx43. Cx40(-/-)Cx43(-/-) mice die around embryonic day 12.5 (E12.5), much earlier than either Cx40(-/-) or Cx43(-/-) mice, and they exhibit malformed hearts with ventricles that are abnormally rotated, suggesting a looping defect. Some Cx40(-/-)Cx43(-/-) animals also develop head defects characteristic of exencephaly. In addition, we examined mice lacking both Cx40 and Cx37 and found a high incidence of atrial and ventricular septal defects at birth. These results provide further evidence for the importance of gap junctions in embryonic development. Moreover, ablating different pairs of cardiac connexins results in distinct heart defects, suggesting both common and unique functions for Cx40, Cx43, and Cx37 during cardiac morphogenesis.     

Connexin 32 fused to the green fluorescent protein retains its ability to control the proliferation of thyroid cells

Cell-to-cell exchanges of signaling molecules are thought to be involved in the control of cell proliferation. Connexins, which are encoded by a family of genes expressed in a cell type-specific manner, are considered as tumor suppressors. Thyroid epithelial cells co-express connexin 32 (Cx32) and connexin 43 (Cx43) that form distinct and delocalized gap junctions in vivo. The communication-deficient rat thyroid-derived cell lines, FRTL-5 and FRT, stably transfected with the Cx32 cDNA, have a reduced proliferation rate related to a prolonged G1 cell cycle phase. To determine whether Cx32-gap junctions exert the same regulatory role in vivo, we have undertaken a program of production of transgenic mice over-expressing Cx32 specifically in thyrocytes. To this purpose, we designed a vector in which the Cx32 cDNA was fused to the gene encoding the enhanced green fluorescent protein (EGFP) and placed under the control of a strong and thyroid-specific promoter, the thyroglobulin gene promoter (pTg). In stably transfected FRTL-5 cells, the Cx32/EGFP chimeric protein forms functional gap junction channels and induces the same proliferation slowdown as native Cx32. The pTg-Cx32/EGFP construct should thus allow us to obtain the thyroid-targeted over-expression of Cx32 in the mouse to investigate the involvement of Cx32-gap junctions in thyroid growth, functional activity and propensity to form tumors.     

Connexin 32 Fused to the Green Fluorescent Protein Retains its Ability to Control the Proliferation of Thyroid Cells

Cell-to-cell exchanges of signaling molecules are thought to be involved in the control of cell proliferation. Connexins, which are encoded by a family of genes expressed in a cell type-specific manner, are considered as tumor suppressors. Thyroid epithelial cells co-express connexin 32 (Cx32) and connexin 43 (Cx43) that form distinct and delocalized gap junctions in vivo. The communication-deficient rat thyroid-derived cell lines, FRTL-5 and FRT, stably transfected with the Cx32 cDNA, have a reduced proliferation rate related to a prolonged G1 cell cycle phase. To determine whether Cx32-gap junctions exert the same regulatory role in vivo, we have undertaken a program of production of transgenic mice over-expressing Cx32 specifically in thyrocytes. To this purpose, we designed a vector in which the Cx32 cDNA was fused to the gene encoding the enhanced green fluorescent protein (EGFP) and placed under the control of a strong and thyroid-specific promoter, the thyroglobulin gene promoter (pTg). In stably transfected FRTL-5 cells, the Cx32/EGFP chimeric protein forms functional gap junction channels and induces the same proliferation slowdown as native Cx32. The pTg-Cx32/EGFP construct should thus allow us to obtain the thyroid-targeted over-expression of Cx32 in the mouse to investigate the involvement of Cx32-gap junctions in thyroid growth, functional activity and propensity to form tumors.     

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.     

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.     

Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies

We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.     

Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.     

Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins.

The cellular distribution of connexin40 (Cx40), a newly cloned gap junction structural protein, was examined by immunofluorescence microscopy using two different specific anti-peptide antibodies. Cx40 was detected in the endothelium of muscular as well as elastic arteries in a punctate pattern consistent with the known distribution of gap junctions. However, it was not detected in other cells of the vascular wall. By contrast, Cx43, another connexin present in the cardiovascular system, was not detected in endothelial cells of muscular arteries but was abundant in the myocardium and aortic smooth muscle. We have tested the ability of these connexins to interact functionally. Cx40 was functionally expressed in pairs of Xenopus oocytes and induced the formation of intercellular channels with unique voltage dependence. Unexpectedly, communication did not occur when oocytes expressing Cx40 were paired with those expressing Cx43, although each could interact with a different connexin, Cx37, to form gap junction channels in paired oocytes. These findings indicate that establishment of intercellular communication can be spatially regulated by the selective expression of different connexins and suggest a mechanism that may operate to control the extent of communication between cells.     

Suppression of the transformed phenotype of breast cancer by tropomyosin-1

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cDNAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18 beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization.     

Impaired Cx43 gap junction endocytosis causes morphological and functional defects in zebrafish

Gap junctions mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting the exchange of molecules, ions, and electrical impulses. Gap junctions are assembled from connexin (Cx) proteins, with connexin 43 (Cx43) being the most ubiquitously expressed and best studied. While the molecular events that dictate the Cx43 life cycle have largely been characterized, the unusually short half-life of Cxs of only 1–5 h, resulting in constant endocytosis and biosynthetic replacement of gap junction channels, has remained puzzling. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256–289), a region known to encode key residues regulating gap junction turnover, is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43^∆256-289) and a zebrafish model (cx43^lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication. Increased Cx43 protein content in cx43^lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques, and longer Cx43 protein half-lives coincide with severely impaired development. Our findings demonstrate for the first time that continuous Cx43 gap junction endocytosis is an essential aspect of gap junction function and, when impaired, gives rise to significant physiological problems as revealed here for cardiovascular development and function.     

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.     

Connexin46 Is Retained as Monomers in a trans-Golgi Compartment of Osteoblastic Cells

Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100–solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.