Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

Connexin 43 (Cx43), the most widely expressed and abundant vertebrate gap junction protein, is phosphorylated at multiple different serine residues during its life cycle. Cx43 is phosphorylated soon after synthesis and phosphorylation changes as it traffics through the endoplasmic reticulum and Golgi to the plasma membrane, ultimately forming a gap junction structure. The electrophoretic mobility of Cx43 changes as the protein proceeds through its life cycle, with prominent bands often labeled P0, P1 and P2. Many reports have indicated changes in “phosphorylation” based on these mobility shifts and others that occur in response to growth factors or other biological effectors. Here, we indicate how phosphospecific and epitope-specific antibodies can be utilized to show when and where certain phosphorylation events occur during the Cx43 life cycle. These reagents show that phosphorylation at S364 and/or S365 is involved in forming the P1 isoform, an event that apparently regulates trafficking to or within the plasma membrane. Phosphorylation at S325, S328 and/or S330 is necessary to form a P2 isoform; and this phosphorylation event is present only in gap junctions. Treatment with protein kinase C activators led to phosphorylation at S368, S279/S282 and S262 with a shift in mobility in CHO, but not MDCK, cells. The shift was dependent on mitogen-activated protein kinase activity but not phosphorylation at S279/S282. However, phosphorylation at S262 could explain the shift. By defining these phosphorylation events, we have begun to sort out the critical signaling pathways that regulate gap junction function.     

Regulation of Connexin43 Gap Junction Protein Triggers Vascular Recovery and Healing in Human Ocular Persistent Epithelial Defect Wounds

Transiently blocking the expression of the gap junction protein connexin43 using antisense oligodeoxynucleotides or blocking hemichannels with connexin mimetic peptides has been shown to significantly improve outcomes in a range of acute wound models. Less is known about their likely effects in nonhealing wounds. In the eye, prolonged inflammation and lack of epithelial recovery in nonhealing corneal epithelial wounds may lead to corneal opacity, blindness or enucleation. We report here the first human applications of antisense oligodeoxynucleotides that transiently block translation of connexin43 in a prospective study of five eyes with severe ocular surface burns (persistent epithelial defects), which were unresponsive to established therapy for 7 days to 8 weeks prior to treatment. Connexin43-specific antisense oligodeoxynucleotide was delivered in cold, thermoreversible Poloxamer407 gel under either an amniotic membrane graft or a bandage contact lens. The connexin43-specific antisense application reduced inflammation within 1–2 days, and in all five eyes complete and stable corneal reepithelialization was obtained. Recovery of the vascular bed and limbal reperfusion appeared to precede corneal epithelial recovery. We conclude that connexin modulation provides a number of benefits for nonhealing ocular burn wounds, one of which is to promote vascular recovery.     

Trafficking and Recycling of the Connexin43 Gap Junction Protein during Mitosis

During the cell cycle, gap junction communication, morphology and distribution of connexin43 (Cx43)‐containing structures change dramatically. As cells round up in mitosis, Cx43 labeling is mostly intracellular and intercellular coupling is reduced. We investigated Cx43 distributions during mitosis both in endogenous and exogenous expressing cells using optical pulse‐chase labeling, correlated light and electron microscopy, immunocytochemistry and biochemical analysis. Time‐lapse imaging of green fluorescent protein (GFP)/tetracysteine tagged Cx43 (Cx43‐GFP‐4C) expressing cells revealed an early disappearance of gap junctions, progressive accumulation of Cx43 in cytoplasmic structures, and an unexpected subset pool of protein concentrated in the plasma membrane surrounding the midbody region in telophase followed by rapid reappearance of punctate plaques upon mitotic exit. These distributions were also observed in immuno‐labeled endogenous Cx43‐expressing cells. Photo‐oxidation of ReAsH‐labeled Cx43‐GFP‐4C cells in telophase confirmed that Cx43 is distributed in the plasma membrane surrounding the midbody as apparent connexons and in cytoplasmic vesicles. We performed optical pulse‐chase labeling and single label time‐lapse imaging of synchronized cells stably expressing Cx43 with internal tetracysteine domains through mitosis. In late telophase, older Cx43 is segregated mainly to the plasma membrane while newer Cx43 is intracellular. This older population nucleates new gap junctions permitting rapid resumption of communication upon mitotic exit.     

Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 µ M forskolin, whereas that of P₀ increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P₀ expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P₀, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

Role of Gap Junction Protein Connexin43 in Astrogliosis Induced by Brain Injury

Astrogliosis is a process that involves morphological and biochemical changes associated with astrocyte activation in response to cell damage in the brain. The upregulation of intermediate filament proteins including glial fibrillary acidic protein (GFAP), nestin and vimentin are often used as indicators for astrogliosis. Although connexin43 (Cx43), a channel protein widely expressed in adult astrocytes, exhibits enhanced immunoreactivity in the peri-lesion region, its role in astrogliosis is still unclear. Here, we correlated the temporal and spatial expression of Cx43 to the activation of astrocytes and microglia in response to an acute needle stab wound in vivo. We found large numbers of microglia devoid of Cx43 in the needle wound at 3 days post injury (dpi) while reactive astrocytes expressing Cx43 were present in the peripheral zone surrounding the injury site. A redistribution of Cx43 to the needle site, corresponding to the increased presence of GFAP-positive reactive astrocytes in the region, was only apparent from 6 dpi and sustained until at least 15 dpi. Interestingly, the extent of microglial activation and subsequent astrogliosis in the brain of Cx43 knockout mice was significantly larger than those of wild type, suggesting that Cx43 expression limits the degree of microgliosis. Although Cx43 is not essential for astrogliosis and microglial activation induced by a needle injury, our results demonstrate that Cx43 is a useful marker for injury induced astrogliosis due to its enhanced expression specifically within a small region of the lesion for an extended period. As a channel protein, Cx43 is a potential in vivo diagnostic tool of asymptomatic brain injury.     

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.     

Selective Effect of PDGF on Connexin43 versus Connexin40 Comprised Gap Junction Channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g_jand γ_j, respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g_jin Cx40/Cx40 pairs, but decreased g_jin the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g_jsuggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g_jinvolved a decrease in both γ_jand Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

Selective Effect of PDGF on Connexin43 versus Connexin40 Comprised Gap Junction Channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g_j and γ_j, respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g_j in Cx40/Cx40 pairs, but decreased g_j in the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g_j suggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g_j involved a decrease in both γ_j and Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

Monovalent Ion Selectivity Sequences of the Rat Connexin43 Gap Junction Channel

The relative permeability sequences of the rat connexin 43 (rCx43) gap junction channel to seven cations and chloride were examined by double whole cell patch clamp recording of single gap junction channel currents in rCx43 transfected neuroblastoma 2A (N2A) cell pairs. The measured maximal single channel slope conductances (γ_j, in pS) of the junctional current-voltage relationships in 115 mM XCl were RbCl (103) ≥ CsCl (102) > KCl (97) > NaCl (79) ≥ LiCl (78) > TMACl (65) > TEACl (53) and for 115 mM KY were KBr (105) > KCl (97) > Kacetate (77) > Kglutamate (61). The single channel conductance-aqueous mobility relationships for the test cations and anions were linear. However, the predicted minimum anionic and cationic conductances of these plots did not accurately predict the rCx43 channel conductance in 115 mM KCl. Instead, the conductance of the rCx43 channel in 115 mM KCl was accurately predicted from cationic and anionic conductance-mobility plots by applying a mobility scaling factor D_x/D_o, which depends upon the relative radii of the permeant ions to an estimated pore radius. Relative permeabilities were determined for all of the monovalent cations and anions tested from asymmetric salt reversal potential measurements and the Goldman-Hodgkin-Katz voltage equation. These experiments estimate the relative chloride to potassium permeability to be 0.13. The relationship between the relative cation permeability and hydrated radius was modeled using the hydrodynamic equation assuming a pore radius of 6.3 ± 0.4 Å. Our data quantitatively demonstrate that the rCx43 gap junction channel is permeable to monovalent atomic and organic cations and anions and the relative permeability sequences are consistent with an Eisenman sequence II or I, respectively. These predictions about the rCx43 channel pore provide a useful basis for future investigations into the structural determinants of the conductance and permeability properties of the connexin channel pore.     

Connexin43 Cardiac Gap Junction Remodeling: Lessons from Genetically Engineered Murine Models

Sudden cardiac death is responsible for several hundred thousand deaths each year in the United States. Multiple lines of evidence suggest that perturbation of gap junction expression and function in the heart, or what has come to be known as cardiac gap junction remodeling, plays a key mechanistic role in the pathophysiology of clinically significant cardiac arrhythmias. Here we review recent studies from our laboratory using genetically engineered murine models to explore mechanisms implicated in pathologic gap junction remodeling and their proarrhythmic consequences, with a particular focus on aberrant posttranslational phosphorylation of connexin43.     

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.     

Conditional Gene Targeting of Connexin43: Exploring the Consequences of Gap Junction Remodeling in the Heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.     

Prognostic Impact of Reduced Connexin43 Expression and Gap Junction Coupling of Neoplastic Stromal Cells in Giant Cell Tumor of Bone

Missense mutations of the GJA1 gene encoding the gap junction channel protein connexin43 (Cx43) cause bone malformations resulting in oculodentodigital dysplasia (ODDD), while GJA1 null and ODDD mutant mice develop osteopenia. In this study we investigated Cx43 expression and channel functions in giant cell tumor of bone (GCTB), a locally aggressive osteolytic lesion with uncertain progression. Cx43 protein levels assessed by immunohistochemistry were correlated with GCTB cell types, clinico-radiological stages and progression free survival in tissue microarrays of 89 primary and 34 recurrent GCTB cases. Cx43 expression, phosphorylation, subcellular distribution and gap junction coupling was also investigated and compared between cultured neoplastic GCTB stromal cells and bone marow stromal cells or HDFa fibroblasts as a control. In GCTB tissues, most Cx43 was produced by CD163 negative neoplastic stromal cells and less by CD163 positive reactive monocytes/macrophages or by giant cells. Significantly less Cx43 was detected in α-smooth muscle actin positive than α-smooth muscle actin negative stromal cells and in osteoclast-rich tumor nests than in the adjacent reactive stroma. Progressively reduced Cx43 production in GCTB was significantly linked to advanced clinico-radiological stages and worse progression free survival. In neoplastic GCTB stromal cell cultures most Cx43 protein was localized in the paranuclear-Golgi region, while it was concentrated in the cell membranes both in bone marrow stromal cells and HDFa fibroblasts. In Western blots, alkaline phosphatase sensitive bands, linked to serine residues (Ser369, Ser372 or Ser373) detected in control cells, were missing in GCTB stromal cells. Defective cell membrane localization of Cx43 channels was in line with the significantly reduced transfer of the 622 Da fluorescing calcein dye between GCTB stromal cells. Our results show that significant downregulation of Cx43 expression and gap junction coupling in neoplastic stromal cells are associated with the clinical progression and worse prognosis in GCTB.     

Connexin43 PDZ2 Binding Domain Mutants Create Functional Gap Junctions and Exhibit Altered Phosphorylation

Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43ΔI382) or the last five amino acids (Cx43Δ378-382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43ΔI382 and Cx43Δ378-382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.     

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.     

Influence of V5/6-His Tag on the Properties of Gap Junction Channels Composed of Connexin43, Connexin40 or Connexin45

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.     

间隙连接功能多样性:连接蛋白基因敲除研究

编码细胞间间隙连接通道结构蛋白的基因是称为连接蛋白(connexin,Cx)基因的多基因家族;间隙连接蛋白基因有20种,且多数细胞同时表达多种连接蛋白,其功能研究较为复杂;小鼠7种连接蛋白基因的敲除为研究连接蛋白功能多样性提供了良好模型,并揭示了不同连接蛋白在维持不同组织正常发育和代谢中的重要作用.