Gap junction proteins in the light-damaged albino rat.
ABSTRACT: PURPOSE: Changes in connexin expression are associated with many pathological conditions seen in animal models and in humans. We hypothesized that gap junctions are important mediators in tissue dysfunction and injury processes in the retina, and therefore, we investigated the pattern of connexin protein expression in the light-damaged albino rat eye. METHODS: Adult Sprague-Dawley rats were exposed to intense light for 24 h. The animals were euthanized, and ocular tissue was harvested at 0 h, 6 h, 24 h, 48 h, and 7 days after light damage. The tissues were processed for immunohistochemistry and western blotting to analyze the expression of the gap junction proteins in the light-damaged condition compared to the non-light-damaged condition. Cell death was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. RESULTS: Intense light exposure caused increased TUNEL labeling of photoreceptor cells. Immunocytochemistry revealed that connexin 36 (Cx36) was significantly increased in the inner plexiform layer and Cx45 was significantly decreased in the light-damaged retina. The pattern of Cx36 and Cx45 labeling returned to normal 7 days after light damage. Cx43 significantly increased in the RPE and the choroid in the light-damaged tissue, and decreased but not significantly in the retina. This elevated Cx43 expression in the choroid colocalized with markers of nitration-related oxidative stress (nitrotyrosine) and inflammation (CD45 and ionized calcium-binding adaptor molecule-1) in the choroid. CONCLUSIONS: The results suggest that connexins are regulated differently in the retina than in the choroid in response to photoreceptor damage. Changes in connexins, including Cx36, Cx43, and Cx45, may contribute to the damage process. Specifically, Cx43 was associated with inflammatory damage. Therefore, connexins may be candidate targets for treatment for ameliorating disease progression.
Project description:Odorant/receptor binding and initial olfactory information processing occurs in olfactory receptor neurons (ORNs) within the olfactory epithelium. Subsequent information coding involves high-frequency spike synchronization of paired mitral/tufted cell dendrites within olfactory bulb (OB) glomeruli via positive feedback between glutamate receptors and closely-associated gap junctions. With mRNA for connexins Cx36, Cx43 and Cx45 detected within ORN somata and Cx36 and Cx43 proteins reported in ORN somata and axons, abundant gap junctions were proposed to couple ORNs. We used freeze-fracture replica immunogold labeling (FRIL) and confocal immunofluorescence microscopy to examine Cx36, Cx43 and Cx45 protein in gap junctions in olfactory mucosa, olfactory nerve and OB in adult rats and mice and early postnatal rats. In olfactory mucosa, Cx43 was detected in gap junctions between virtually all intrinsic cell types except ORNs and basal cells; whereas Cx45 was restricted to gap junctions in sustentacular cells. ORN axons contained neither gap junctions nor any of the three connexins. In OB, Cx43 was detected in homologous gap junctions between almost all cell types except neurons and oligodendrocytes. Cx36 and, less abundantly, Cx45 were present in neuronal gap junctions, primarily at "mixed" glutamatergic/electrical synapses between presumptive mitral/tufted cell dendrites. Genomic analysis revealed multiple miRNA (micro interfering RNA) binding sequences in 3'-untranslated regions of Cx36, Cx43 and Cx45 genes, consistent with cell-type-specific post-transcriptional regulation of connexin synthesis. Our data confirm absence of gap junctions between ORNs, and support Cx36- and Cx45-containing gap junctions at glutamatergic mixed synapses between mitral/tufted cells as contributing to higher-order information coding within OB glomeruli.
Project description:Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) are a common feature in mammalian brain circuitry, but less is known about their deployment in spinal cord. It has been reported based on connexin mRNA and/or protein detection that developing and/or mature motoneurons express a variety of connexins, including Cx26, Cx32, Cx36 and Cx43 in trigeminal motoneurons, Cx36, Cx37, Cx40, Cx43 and Cx45 in spinal motoneurons, and Cx32 in sexually dimorphic motoneurons. We re-examined the localization of these connexins during postnatal development and in adult rat and mouse using immunofluorescence labeling for each connexin. We found Cx26 in association only with leptomeninges in the trigeminal motor nucleus (Mo5), Cx32 only with oligodendrocytes and myelinated fibers among motoneurons in this nucleus and in the spinal cord, and Cx37, Cx40 and Cx45 only with blood vessels in the ventral horn of spinal cord, including those among motoneurons. By freeze-fracture replica immunolabeling, > 100 astrocyte gap junctions but no neuronal gap junctions were found based on immunogold labeling for Cx43, whereas 16 neuronal gap junctions at postnatal day (P)4, P7 and P18 were detected based on Cx36 labeling. Punctate labeling for Cx36 was localized to the somatic and dendritic surfaces of peripherin-positive motoneurons in the Mo5, motoneurons throughout the spinal cord, and sexually dimorphic motoneurons at lower lumbar levels. In studies of electrical synapses and electrical transmission between developing and between adult motoneurons, our results serve to focus attention on mediation of this transmission by gap junctions composed of Cx36.
Project description:Mammalian retinas contain abundant neuronal gap junctions, particularly in the inner plexiform layer (IPL), where the two principal neuronal connexin proteins are Cx36 and Cx45. Currently undetermined are coupling relationships between these connexins and whether both are expressed together or separately in a neuronal subtype-specific manner. Although Cx45-expressing neurons strongly couple with Cx36-expressing neurons, possibly via heterotypic gap junctions, Cx45 and Cx36 failed to form functional heterotypic channels in vitro. We now show that Cx36 and Cx45 coexpressed in HeLa cells were colocalized in immunofluorescent puncta between contacting cells, demonstrating targeting/scaffolding competence for both connexins in vitro. However, Cx36 and Cx45 expressed separately did not form immunofluorescent puncta containing both connexins, supporting lack of heterotypic coupling competence. In IPL, 87% of Cx45-immunofluorescent puncta were colocalized with Cx36, supporting either widespread heterotypic coupling or bihomotypic coupling. Ultrastructurally, Cx45 was detected in 9% of IPL gap junction hemiplaques, 90-100% of which also contained Cx36, demonstrating connexin coexpression and cotargeting in virtually all IPL neurons that express Cx45. Moreover, double replicas revealed both connexins in separate domains mirrored on both sides of matched hemiplaques. With previous evidence that Cx36 interacts with PDZ1 domain of zonula occludens-1 (ZO-1), we show that Cx45 interacts with PDZ2 domain of ZO-1, and that Cx36, Cx45, and ZO-1 coimmunoprecipitate, suggesting that ZO-1 provides for coscaffolding of Cx45 with Cx36. These data document that in Cx45-expressing neurons of IPL, Cx45 is almost always accompanied by Cx36, forming "bihomotypic" gap junctions, with Cx45 structurally coupling to Cx45 and Cx36 coupling to Cx36.
Project description:Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies.
Project description:The gap junction protein connexin36 (CX36) has been well studied in the mature central nervous system, but there has been little information regarding its possible roles in embryonic development. We report here the isolation of the full-length chick CX36 coding sequence (predicted M(r) 35.1 kDa) and its strikingly restricted pattern of gene expression in the mesoderm of the chick embryo. In situ hybridization experiments demonstrated CX36 expression in somites by embryonic day 2. The transcripts first appeared dorsomedially within the somite and expanded ventrolaterally to form stripes in the middle of each somite. The CX36 stripes fell within somitic territories enriched in MYOD and FGF8 expression and impoverished in PAX3 transcripts, establishing that CX36 mRNA is expressed in the myotome. We compared the somitic expression pattern of CX36 with those of three other connexins, CX42, CX43, and CX45. At embryonic day 4, CX42 transcripts were localized to the myotome in a pattern resembling that of CX36. In contrast, CX43 was enriched in the dermomyotome, and CX45 was detected in both the myotome and the dermomyotome. Immunoblotting using Cx36 antibodies demonstrated bands of identical electrophoretic mobilities in trunk and retinal homogenates, and Cx36 immunostaining detected punctate immunoreactivity in the myotome. These results demonstrate that some connexins in the developing mesoderm are broadly expressed whereas others are highly localized, and suggest that CX36, CX42, and CX45 are involved in intercellular communication among developing muscle cells.
Project description:Connexins are a family of transmembrane proteins that form gap junction channels. These proteins undergo both proteasomal and lysosomal degradation, mechanisms that serve to regulate connexin levels. Our previous work described CIP75 [connexin43 (Cx43)-interacting protein of 75 kDa], a protein involved in proteasomal degradation, as a novel Cx43-interacting protein. We have discovered two additional connexins, connexin40 (Cx40) and connexin45 (Cx45), that interact with CIP75. Nuclear magnetic resonance (NMR) analyses identified the direct interaction of the CIP75 UBA domain with the carboxyl-terminal (CT) domains of Cx40 and Cx45. Reduction in CIP75 by shRNA in HeLa cells expressing Cx40 or Cx45 resulted in increased levels of the connexins. Furthermore, treatment with trafficking inhibitors confirmed that both connexins undergo endoplasmic reticulum-associated degradation (ERAD), and that CIP75 preferentially interacts with the connexin proteins bound for proteasomal degradation from the ER. In addition, we have also discovered that CIP75 interacts with ER-localized Cx32 in a process that is likely mediated by Cx32 ubiquitination. Thus, we have identified novel interacting connexin proteins of CIP75, indicating a role for CIP75 in regulating the levels of connexins in general, through proteasomal degradation.
Project description:BACKGROUND: Gap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues. Each channel is composed of 12 protein subunits, termed connexins (Cx). In the mouse retina, Cx43 could be localized mostly between astroglial cells whereas expression of Cx36, Cx45 and Cx57 genes has been detected in different neuronal subtypes. In the human retina, however, the expression pattern of connexin genes is largely unknown. METHODS: Northern blot hybridizations, RT-PCR as well as immunofluorescence analyses helped to explore at least partially the expression pattern of the following human connexin genes GJD2 (hCx36), GJC1 (hCx45), GJA9 (hCx59) and GJA10 (hCx62) in the human retina. RESULTS: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA. Immunofluorescence signals for both connexins could be located in both inner and outer plexiform layer (IPL, OPL). Expression of a third connexin gene denoted as GJA10 (Cx62) was also detected after Northern blot hybridization in the human retina. Interestingly, its gene structure is similar to that of Gja10 (mCx57) being expressed in mouse horizontal cells. RT-PCR analysis suggested that an additional exon of about 25 kb further downstream, coding for 12 amino acid residues, is spliced to the nearly complete reading frame on exon2 of GJA10 (Cx62). Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina. CONCLUSION: In contrast to the neuron-expressed connexin genes Gjd2 coding for mCx36, Gjc1 coding for mCx45 and Gja10 coding for mCx57 in the mouse, a subset of 4 connexin genes, including the unique GJA9 (Cx59) and GJA10 (Cx62), could be detected at least as transcript isoforms in the human retina. First immunofluorescence analyses revealed a staining pattern of hCx36 and hCx45 expression both in the IPL and OPL, partially reminiscent to that in the mouse, although additional post-mortem material is needed to further explore their sublamina-specific distribution. Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.
Project description:Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells.We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication.Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.
Project description:In cardiac tissues, the expression of multiple connexins (Cx40, Cx43, Cx45, and Cx30.2) is a requirement for proper development and function. Gap junctions formed by these connexins have distinct permeability and gating mechanisms. Since a single cell can express more than one connexin isoform, the formation of hetero-multimeric gap junction channels provides a tissue with an enormous repertoire of combinations to modulate intercellular communication. To study further the perm-selectivity and gating properties of channels containing Cx43 and Cx45, we studied two monoheteromeric combinations in which a HeLa cell co-transfected with Cx43 and Cx45 was paired with a cell expressing only one of these connexins. Macroscopic measurements of total conductance between cell pairs indicated a drastic reduction in total conductance for mono-heteromeric channels. In terms of Vj dependent gating, Cx43 homomeric connexons facing heteromeric connexons only responded weakly to voltage negativity. Cx45 homomeric connexons exhibited no change in Vj gating when facing heteromeric connexons. The distributions of unitary conductances (?j) for both mono-heteromeric channels were smaller than predicted, and both showed low permeability to the fluorescent dyes Lucifer yellow and Rhodamine123. For both mono-heteromeric channels, we observed flux asymmetry regardless of dye charge: flux was higher in the direction of the heteromeric connexon for MhetCx45 and in the direction of the homomeric Cx43 connexon for MhetCx43. Thus, our data suggest that co-expression of Cx45 and Cx43 induces the formation of heteromeric connexons with greatly reduced permeability and unitary conductance. Furthermore, it increases the asymmetry for voltage gating for opposing connexons, and it favors asymmetric flux of molecules across the junction that depends primarily on the size (not the charge) of the crossing molecules.
Project description:HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V(j)-sensitive gating of I(j) (V(j), gap junction voltage; I(j), gap junction current), (2) contribution and (3) kinetics of I(j) deactivation and (4) single-channel conductance. The first three reflect alterations of fast V(j) gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.