The cataract causing Cx50-S50P mutant inhibits Cx43 and intercellular communication in the lens epithelium

Mutations in Connexin50 (Cx50) cause cataracts in both humans and mice. The mechanism(s) behind how mutated connexins lead to a variety of cataracts have yet to be fully elucidated. Here, we tested whether the cataract inducing Cx50-S50P mutant interacts with wild-type Connexin43 (Cx43) to form mixed channels with attenuated function. Using dual whole-cell voltage clamp, immunofluorescent microscopy and in situ dye transfer analysis we identified a unique interaction between the mutant subunit and wild-type Cx43. In paired Xenopus oocytes, co-expression of Cx50-S50P with Cx43 reduced electrical coupling ≥ 90%, without a reduction in protein expression. In transfected cells, Cx50-S50P did not target to cell-cell interfaces by itself, but co-expression of Cx50-S50P with Cx43 resulted in its localization at areas of cell-cell contact. We used Cx43 conditional knockout, Cx50 knockout and Cx50-S50P mutant mice to examine this interaction in vivo. Mice expressing both Cx43 and Cx50-S50P in the lens epithelium revealed a unique expression pattern for Cx43 and a reduction in Cx43 protein. In situ dye transfer experiments showed that the Cx50-S50P mutant, but not the Cx50, or Cx43 conditional knockout, greatly inhibited epithelial cell gap junctional communication in a manner similar to a double knockout of Cx43 and Cx50. The inhibitory affects of Cx50-S50P lead to diminished electrical coupling in vitro, as well as a discernable reduction in epithelial cell dye permeation. These data suggest that dominant inhibition of Cx43 mediated epithelial cell coupling may play a role in the lens pathophysiology caused by the Cx50-S50P mutation.     

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size.     

Internal Ribosomal Entry Site (IRES) Activity Generates Endogenous Carboxyl-terminal Domains of Cx43 and Is Responsive to Hypoxic Conditions

Connexin43 (Cx43) is the most abundant gap junction protein in higher vertebrate organisms and has been shown to be involved in junctional and non-junctional functions. In addition to the expression of full-length Cx43, endogenously produced carboxyl-terminal segments of Cx43 have been described and have been suggested to be involved in manifold biological functions, such as hypoxic preconditioning and neuronal migration. Molecular aspects, however, behind the separate generation of carboxyl-terminal segments of Cx43 have remained elusive. Here we report on a mechanism that may play a key role in the separate production of these domains. First, stringent evidence derived from siRNA treatment and specific knockouts revealed significant loss of the low molecular weight fragments of Cx43. By applying a dicistronic vector strategy on transfected cell lines, we were able to identify putative IRES activity (nucleotides 442–637) in the coding region of Cx43, which resides upstream from the nucleotide sequence encoding the carboxyl terminus (nucleotides 637–1149). Functional responsiveness of the endogenous expression of Cx43 fragments to hypoxic/ischemic treatment was evaluated in in vitro and in vivo models, which led to a significant increase of the fastest migrating form (20 kDa) under conditions of metabolic deprivation. By nano-MS spectrometry, we achieved stringent evidence of the identity of the 20-kDa segment as part of the carboxyl-terminal domain of full-length Cx43. Our data prove the existence of endogenously expressed carboxyl-terminal domains, which may serve as valuable tools for further translational application in ischemic disorders.     

Increasing Gap Junctional Coupling: A Tool for Dissecting the Role of Gap Junctions

Much of our current knowledge about the physiological and pathophysiological role of gap junctions is based on experiments where coupling has been reduced by either chemical agents or genetic modification. This has brought evidence that gap junctions are important in many physiological processes. In a number of cases, gap junctions have been implicated in the initiation and progress of disease, and experimental uncoupling has been used to investigate the exact role of coupling. The inverse approach, i.e., to increase coupling, has become possible in recent years and represents a new way of testing the role of gap junctions. The aim of this review is to summarize the current knowledge obtained with agents that selectively increase gap junctional intercellular coupling. Two approaches will be reviewed: increasing coupling by the use of antiarrhythmic peptide and its synthetic analogs and by interfering with the gating of gap junctional channels.     

Divalent regulation and intersubunit interactions of human Connexin26 (Cx26) hemichannels

Control of plasma membrane connexin hemichannel opening is indispensable, and is achieved by physiological extracellular divalent ion concentrations. Here, we explore the differences between regulation by Ca²⁺ and Mg²⁺ of human connexin26 (hCx26) hemichannels and the role of a specific interaction in regulation by Ca²⁺. To effect hemichannel closure, the apparent affinity of Ca²⁺ (0.33 mM) is higher than for Mg²⁺ (1.8 mM). Hemichannel closure is accelerated by physiological Ca²⁺ concentrations, but non-physiological concentrations of extracellular Mg²⁺ are required for this effect. Our recent report provided evidence that extracellular Ca²⁺ facilitates hCx26 hemichannel closing by disrupting a salt bridge interaction between positions D50 and K61 that stabilizes the open state. New evidence from mutant cycle analysis indicates that D50 also interacts with Q48. We find that the D50-Q48 interaction contributes to stabilization of the open state, but that it is relatively insensitive to disruption by extracellular Ca²⁺ compared with the D50-K61 interaction.     

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.     

Gap Junction Intercellular Communication Mediated by Connexin43 in Astrocytes Is Essential for Their Resistance to Oxidative Stress

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H₂O₂) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H₂O₂ transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H₂O₂-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H₂O₂ insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H₂O₂ treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.     

Amino Acid Residue Val362 Plays a Critical Role in Maintaining the Structure of C Terminus of Connexin 50 and in Lens Epithelial-fiber Differentiation

We have previously shown that connexin (Cx) 50, unlike the other two lens connexins, Cx43 and Cx46, promotes chicken lens epithelial-fiber differentiation in a channel-independent manner. Here, we show that deletion of the PEST motif at the C terminus (CT) domain of Cx50 attenuates the stimulatory effect of Cx50 on lens fiber differentiation. Valine 362, a residue located within the PEST domain, is functionally involved. The structure of the Cx50 CT predicted by molecular modeling revealed four α-helices and Val³⁶² was found to be located in the middle of the 3rd helix. Replacement of Val³⁶² with amino acid residues that disrupt the α-helical structure predicted by molecular modeling, such as arginine, glutamate, or phenylalanine, attenuated the stimulatory effects of Cx50 on lens differentiation, whereas replacement with threonine, isoleucine, leucine, or proline, which maintain the structure preserved the function of Cx50. Circular dichroism (CD) studies supported the structural predictions and showed that the substitution with Glu, but not Thr or Pro, disrupted the α-helix, which appears to be the structural feature important for lens epithelial-fiber differentiation. Together, our results suggest that Val³⁶² is important for maintaining the helical structure and is crucial for the role of Cx50 in promoting lens epithelial-fiber differentiation.     

Histological alterations in the livers of Cx43-deficient mice submitted to a cholestasis model

Gap junction intercellular communication capacity and connexin expression are reportedly involved in cell proliferation. To understand the participation of connexins in biliary duct hyperplasia, a cholestasis model was applied to mice with heterologous deletion of Gja 1, the connexin 43 (Cx43) gene. Heterozygous (Cx43+/-) knockout (KO) and wild-type mice (Cx43+/+) (WT) were submitted to bile duct ligation and euthanized at different time points (48 h, 7 days, and 14 days) after surgery. After euthanasia, the macroscopic and microscopic liver alterations were examined. A histomorphometric study of the livers was performed. For this purpose, a grid containing 100 points was applied to each liver section. The volumetric fraction of bile ducts, hepatocytes, arterioles, and terminal hepatic vein were quantified. Cell proliferation was also quantified by western blot PCNA. High mortality was observed in both genotypes. The heterologous deletion of Cx43 did not affect the biliary duct hyperplasia or most of the other parameters analyzed; however, the Cx43-deficient mice showed decrease in hepatic vein angiogenesis in comparison with the wild-type mice 48 h after surgery. In conclusion, our results indicate that the Cx43 gene heterologous deletion does not affect the biliary duct hyperplasia; on the other hand, connexin 43 deficiencies do affect the hepatic vein angiogenesis, although other studies to understand the details of this influence will be necessary.     

Connexin43 with a cytoplasmic loop deletion inhibits the function of several connexins

Connexins (Cx) form gap junction channels mediating direct intercellular communication. To study the role of amino acids within the cytoplasmic loop, we produced a recombinant adenovirus containing Cx43 with a deletion of amino acids 130-136 (Cx43del(130-136)). Cx43del(130-136) expressed alone in HeLa cells localized within the cytoplasm and did not allow transfer of ions, neurobiotin or Lucifer yellow. When co-expressed with wild type Cx43, Cx43del(130-136) blocked electrical coupling and transfer of neurobiotin or Lucifer yellow. Cx43del(130-136) and Cx43 co-localized by immunofluorescence and were co-purified from Triton X-100-solubilized cell extracts. Intercellular transfer mediated by Cx37 and Cx45 (but not Cx26 or Cx40) was inhibited when co-expressed with Cx43del(130-136). Cx43del(130-136) co-localized with Cx37, Cx40, or Cx45, but not Cx26. These data suggest that Cx43del(130-136) produces connexin-specific inhibition of intercellular communication through formation of heteromeric connexons that are non-functional and/or retained in the cytoplasm.     

Phosphorylation in the C-terminus of the rat connexin46 (rCx46) and regulation of the conducting activity of the formed connexons

To analyse the role of PKC-dependent phosphorylation in the C-terminus of rCx46 in regulation of rCx46 connexons, truncated mutants rCx46_45.3 and rCx46_44.2 which end before and after PKC-dependent phosphorylation sites respectively were generated. Both rCx46_45.3 and rCx46_44.2 formed connexons in Xenopus oocytes similar to Cx46_wt-connexons. They were activated by depolarisation above −40 mV and at voltages above 50 mV, inactivation was spontaneously observed or induced by PKC activator TPA, suggesting that inactivation does not require PKC-dependent phosphorylation in the C-terminus. Three casein-kinase-II-(CKII)-dependent phosphorylation sites were also identified. rCx46_37.7 and rCx46_28.2 respectively without two or all of these sites were generated. rCx46_37.7-connexons were similar to rCx46_wt-connexons. rCx46_28.2-connexons comparable to rCx46_wt-connexons were observed after injection of 50 times more rCx46_28.2-mRNA (25 ng per oocyte). CKII-blocker inhibited depolarisation-evoked currents in oocytes injected with 0.5 ng per oocyte rCx46_37.7-mRNA or rCx46_wt-mRNA. Injection of 25 ng per oocyte rCx46_37.7-mRNA or rCx46_wt-mRNA overcame the effect of CKII-inhibitor. We propose that CKII-dependent phosphorylation in the C-terminus accelerates formation of rCx46-connexons.     

Connexin 26 (GJB2) mutations, causing KID Syndrome, are associated with cell death due to calcium gating deregulation

The autosomic dominant KID Syndrome (MIM 148210), due to mutations in GJB2 (connexin 26, Cx26), is an ectodermal dysplasia with erythematous scaly skin lesions, keratitis and severe bilateral sensorineural deafness. The Cx26 protein is a component of gap junction channels in epithelia, including the cochlea, which coordinates the exchange of molecules and ions. Here, we demonstrate that different Cx26 mutants (Cx26D50N and Cx26G11E) cause cell death in vitro by the alteration of intra-cellular calcium concentrations. These results help to explain the pathogenesis of both the hearing and skin phenotypes, since calcium is also a potent regulator of the epidermal differentiation process.     

Quantitative Determination of Gap Junctional Permeability in the Lens Cortex

We have developed a simple dye transfer method, which allows the gap junction permeability of lens fiber cells to be quantified. Two fixable fluorescent dyes (Lucifer yellow and rhodamine-dextran) were introduced into peripheral lens fiber cells via mechanical damage induced by removing the lens capsule. After a defined incubation period, lenses were fixed, sectioned, and the distribution of the dye recorded using confocal microscopy. Rhodamine-dextran and Lucifer yellow both labeled the extracellular space between fiber cells and the cytoplasm of fiber cells that had been damaged by capsule removal. For the gap junctional permeable dye Lucifer yellow, however, labeling was not confined to the damaged cells and exhibited intercellular diffusion away from the damaged cells. The extent of dye diffusion was quantified by collecting radial dye intensity profiles from the confocal images. Effective diffusion coefficients (D _eff ) for Lucifer yellow were then calculated by fitting the profiles to a series of model equations, which describe radial diffusion in a sphere. D _eff is the combination of dye diffusion through the cytoplasm and through gap junction channels. To calculate the gap junctional permeability (P _j ) an estimate of the cytoplasmic diffusion coefficient (D _cyt = 0.7 × 10⁻⁶ cm²/sec) was obtained by observing the time course of dye diffusion in isolated elongated fiber cells loaded with Lucifer yellow via a patch pipette. Using this approach, we have obtained a value for P _j of 31 × 10⁻⁵ cm/sec for fiber-fiber gap junctions. This value is significantly larger than the value of P _j of 4.4 × 10⁻⁶ cm/sec reported by Rae and coworkers for epithelial-fiber junctions (Rae et al., 1996. J. Membrane Biol. 150:89–103), and most likely reflects the high abundance of gap junctions between lens fiber cells. Received: 1 December 1998/Revised: 22 February 1999     

Identification of the Binding Site of Methylglyoxal on Glutathione Peroxidase: Methylglyoxal Inhibits Glutathione Peroxidase Activity via Binding to Glutathione Binding Sites Arg 184 and 185

Methylglyoxal (MG), a physiological &#102 -dicarbonyl compound is derived from glycolytic intermediates and produced during the Maillard reaction. The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino group of proteins, contributes to the aging of proteins and to complications associated with diabetes. In our previous studies (Che, et al. (1997) "Selective induction of heparin-binding epidermal growth factor-like growth factor by MG and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes". J. Biol. Chem., 272 , 18453-18459), we reported that MG elevates intracellular peroxide levels, but the mechanisms for this remain unclear. Here, we report that MG inactivates bovine glutathione peroxidase (GPx), a major antioxidant enzyme, in a dose- and time-dependent manner. The use of BIAM labeling, it was showed that the selenocysteine residue in the active site was intact when GPx was incubated with MG. MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and protein sequencing examined the possibility that MG modifies arginine residues in GPx. The results show that Arg 184 and Arg 185, located in the glutathione binding site of GPx was irreversively modified by treatment with MG. Reactive dicarbonyl compounds such as 3-deoxyglucosone, glyoxal and phenylglyoxal also inactivated GPx, although the rates for this inactivation varied widely. These data suggest that dicarbonyl compounds are able to directly inactivate GPx, resulting in an increase in intracellular peroxides which are responsible for oxidative cellular damage.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Role of the p38 MAP-Kinase Signaling Pathway for Cx32 and Claudin-1 in the Rat Liver

Liver regeneration and cholestasis are associated with adaptive changes in expression of gap and tight junctions through signal transduction. The roles of stress responsitive MAP-kinase, p38 MAP-kinase, in the signaling pathway for gap junction protein, Cx32, and tight junction protein, claudin-1, were examined in rat liver in vivoand in vitro, including regeneration following partial hepatectomy and cholestasis after common bile duct ligation. Changes in the expression and function of Cx32 and claudin-1 in hepatocytes in vivowere studied using the p38 MAP-kinase inhibitor SB203580. Following partial hepatectomy and common bile duct ligation, down-regulation of Cx32 protein was inhibited by SB203580 treatment. Up-regulation of claudin-1 protein was enhanced by SB203580 treatment after partial hepatectomy but not common bile duct ligation. However, no change of the Ki-67 labeling index (which is a marker for cell proliferation) in the livers treated with SB203580, was observed compared to that without SB203580 treatment. In primary cultures of rat hepatocytes, however, treatment with a p38 MAP-kinase activator, anisomycin, decreased Cx32 and claudin-1 protein levels. p38 MAP-kinase may be an important signaling pathway for regulation of gap and tight junctions in hepatocytes. Changes of gap and tight junctions during liver regeneration and cholestasis are shown to be in part controlled via the p38 MAP-kinase signaling pathway and are independent of cell growth.