Cytosolic stress reduces degradation of connexin43 internalized from the cell surface and enhances gap junction formation and function

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised.     

Ubiquitination of Gap Junction Proteins

Gap junctions are plasma membrane domains containing arrays of channels that exchange ions and small molecules between neighboring cells. Gap junctional intercellular communication enables cells to directly cooperate both electrically and metabolically. Several lines of evidence indicate that gap junctions are important in regulating cell growth and differentiation and for maintaining tissue homeostasis. Gap junction channels consist of a family of transmembrane proteins called connexins. Gap junctions are dynamic structures, and connexins have a high turnover rate in most tissues. Connexin43 (Cx43), the best-studied connexin isoform, has a half-life of 1.5–5 h; and its degradation involves both the lysosomal and proteasomal systems. Increasing evidence suggests that ubiquitin is important in the regulation of Cx43 endocytosis. Ubiquitination of Cx43 is thought to occur at the plasma membrane and has been shown to be regulated by protein kinase C and the mitogen-activated protein kinase pathway. Cx43 binds to the E3 ubiquitin ligase Nedd4, in a process modulated by Cx43 phosphorylation. The interaction between Nedd4 and Cx43 is mediated by the WW domains of Nedd4 and involves a proline-rich sequence conforming to a PY (XPPXY) consensus motif in the C terminus of Cx43. In addition to the PY motif, an overlapping tyrosine-based sorting signal conforming to the consensus of an YXXϕ motif is involved in Cx43 endocytosis, indicating that endocytosis of gap junctions involves both ubiquitin-dependent and -independent pathways. Here, we discuss current knowledge on the ubiquitination of connexins.     

Dose-dependent differential upregulation of CCN1/Cyr61 and CCN3/NOV by the gap junction protein Connexin43 in glioma cells

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.     

Interactions of connexins with other membrane channels and transporters

Cell-to-cell communication through gap junctions exists in most animal cells and is essential for many important biological processes including rapid transmission of electric signals to coordinate contraction of cardiac and smooth muscle, the intercellular propagation of Ca(2+) waves and synchronization of physiological processes between adjacent cells within a tissue. Recent studies have shown that connexins (Cx) can have either direct or indirect interactions with other plasma membrane ion channels or membrane transport proteins with important functional consequences. For example, in tissues most severely affected by cystic fibrosis (CF), activation of the CF Transmembrane Conductance Regulator (CFTR) has been shown to influence connexin function. Moreover, a direct interaction between Cx45.6 and the Major Intrinsic Protein/AQP0 in lens appears to influence the process of cell differentiation whereas interactions between aquaporin 4 (AQP4) and Cx43 in mouse astrocytes may coordinate the intercellular movement of ions and water between astrocytes. In this review, we discuss evidence supporting interactions between Cx and membrane channels/transporters including CFTR, aquaporins, ionotropic glutamate receptors, and between pannexin1, another class of putative gap-junction-forming proteins, and Kvbeta3, a regulatory beta-subunit of voltage gated potassium channels. Although the precise molecular nature of these interactions has yet to be defined, their consequences may be critical for normal tissue homeostasis.     

The proteasome regulates the interaction between Cx43 and ZO-1

Gap junction (GJ) intercellular communication (GJIC) is vital to ensure proper cell and tissue function. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. In this study we have investigated the role of the proteasome in regulation of connexin 43 (Cx43) internalization. Although the participation of both the proteasome and lysosome has long been suggested in Cx43 degradation, the molecular mechanisms whereby proteasome contributes to regulate Cx43 internalization and intercellular communication are still unclear. The results presented in this study envision a new mechanism whereby proteasome regulates GJIC by modulating interaction between Cx43 and ZO-1. Immunoprecipitation experiments, in the presence of proteasome inhibitors, together with immunofluorescence data indicate that the proteasome regulates interaction between Cx43 and ZO-1. Overexpression of the PDZ2 domain of ZO-1 and the expression of Cx-43 fused in frame with a V5/HIS tag, suggest that interaction between the two proteins occurs through the PDZ2 domain of ZO-1 and the C-terminus of Cx43. When interaction between Cx43 and ZO-1 is reduced, as in the presence of proteasome inhibitors, Cx43 accumulates, forming large GJ plaques at plasma membrane. Data presented in this article suggest a new pathway whereby alterations in proteasome activity may impact on GJIC as well as on non-junctional communication with extracellular environment, contributing to cell and tissue dysfunction.     

Roles and regulation of lens epithelial cell connexins

The avascular lens of the eye is covered anteriorly by an epithelium containing nucleated, metabolically active cells. This epithelium contains the first lens cells to encounter noxious external stimuli and cells that can develop compensatory or protective responses. Lens epithelial cells express the gap junction proteins, connexin43 (Cx43) and connexin50 (Cx50). Cx43 and Cx50 form gap junction channels and hemichannels with different properties. Although they may form heteromeric hemichannels, Cx43 and Cx50 probably do not form heterotypic channels in the lens. Cx50 channels make their greatest contribution to intercellular communication during the early postnatal period; subsequently, Cx43 becomes the predominant connexin supporting intercellular communication. Although epithelial Cx43 appears dispensable for lens development, Cx50 is critical for epithelial cell proliferation and differentiation. Cx43 and Cx50 hemichannels and gap junction channels are regulated by multiple different agents. Lens epithelial cell connexins contribute to both normal lens physiology and pathology.     

Heteromeric Mixing of Connexins: Compatibility of Partners and Functional Consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Heteromeric mixing of connexins: compatibility of partners and functional consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Heteromeric Mixing of Connexins: Compatibility of Partners and Functional Consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Adenoviral delivery of human connexin37 induces endothelial cell death through apoptosis

Gap junction channels formed of connexins directly link the cytoplasm of adjacent cells and have been implicated in intercellular signaling that may regulate the functions of vascular cells. To facilitate connexin manipulation and analysis of their roles in adult endothelial cells, we developed adenoviruses containing the vascular connexins (Cx37, Cx40, and Cx43). We infected cultured human umbilical vein endothelial cells with control or connexin adenoviruses. Connexin expression was verified by immunoblotting and immunofluorescence. Infection with the Cx37 adenovirus (but not control or other connexin adenoviruses) led to a dose-dependent death of the endothelial cells that was partially antagonized by the gap junction blocker alpha-glycyrrhetinic acid and altered the intercellular transfer of Lucifer yellow and neurobiotin. Cell morphology, Annexin V and TUNEL staining, and caspase 3 assays all implicated apoptosis in the cell death. These data suggest that connexin-specific alterations of intercellular communication may modulate endothelial cell growth and death.     

Functional gap junctions accumulate at the immunological synapse and contribute to T cell activation

Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca(2+) responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell-T cell signaling machinery that regulates T cell activation.     

Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision

Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H₂S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H₂S.     

Up-regulation of connexin 43 and gap junctional intercellular communication by Coleusin Factor is associated with growth inhibition in rat osteosarcoma UMR106 cells

Gap junctions, formed by connexin (Cx) family proteins, permit direct exchange of regulatory ions and small signal molecules between neighbouring cells. Gap junctional intercellular communication (GJIC) plays an important role in maintaining the homeostasis and preventing cell transformation. Most of the tumour cells feature deficient or aberrant connexin expression and GJIC level, and restoration of connexin expression and GJIC is correlated with cell growth control. Numerous researches has suggested the possibility of connexins as potential anti-tumour targets for chemoprevention and chemotherapy. We investigated the ability of Coleusin Factor (CF, also named FSK88) to regulate the Cx43 expression and GJIC level in rat osteosarcoma UMR106 cells. The results have demonstrated that CF increased the mRNA and protein expression of Cx43 in both in a dose- and timedependent manner, and concomitant with up-regulation of Cx43, CF treatment up-regulated the diminished GJIC level in UMR106 cells as assayed by dye transfer experiments. In addition, Cx43 distribution at the plasma membrane was also enhanced dramatically by CF treatment. Furthermore, we discovered that CF was potent to inhibit the growth and proliferation of UMR106 cells. These results provide the first evidence that CF can regulate connexin and GJIC, indicating that Cx43 may be a target of CF to exert its anti-tumour effects.     

Control of Intracellular Movement of Connexins by E-Cadherin in Murine Skin Papilloma Cells

The gap junctional intercellular communication-deficient mouse skin papilloma cell line P3/22 expresses Cx43 but not E-cadherin. The E-cadherin gene-transfected cells (P3E1) communicate in a calcium-dependent manner and they were used to study how E-cadherin restores the function of connexins. At low calcium, Cx43 molecules remain in the cytoplasm of P3E1 cells and appear at cell-cell contact areas only in high-calcium medium. While Cx43 is unphosphorylated in P3E1 cells in low-calcium medium, two phosphorylated bands appeared at high calcium. However, when Cx26, which has no C-terminal tail that can undergo phosphorylation, was expressed in P3E1 cells, this connexin also moved to the plasma membrane after the calcium shift and partly colocalized with Cx43, suggesting that C-terminal phosphorylation is not essential for E-cadherin-mediated intracellular transport of connexins. In low calcium, both Cx26 and Cx43 remained and colocalized in the endoplasmic reticulum. As early as 30 min after the shift to high-calcium medium, both Cx43 and Cx26 began to accumulate in the Golgi apparatus. Intracellular movement of connexins to the cytoplasmic membrane at high calcium was effectively blocked by cytochalasin D and brefeldin A. These results suggest that E-cadherin junction formation at high calcium leads to formation of actin cables, which directly or indirectly transport connexins from the cytoplasm to the cell-cell contact membranes via the Golgi apparatus.     

Role of connexin (gap junction) genes in cell growth control and carcinogenesis

Gap junctional intercellular communication (GJIC) is considered to play a key role in the maintenance of tissue independence and homeostasis in multicellular organisms by controlling the growth of GJIC-connected cells. Gap junction channels are composed of connexin molecules and, so far, more than a dozen different connexin genes have been shown to be expressed in mammals. Reflecting the importance of GJIC in various physiological functions, deletion of different connexin genes from mice results in various disorders, including cancers, heart malformation or conduction abnormality, cataract, etc. The possible involvement of aberrant GJIC in abnormal cell growth and carcinogenesis has long been postulated and recent studies in our own and other laboratories have confirmed that expression and function of connexin genes play an important role in cell growth control. Thus, almost all malignant cells show altered homologous and/or heterologous GJIC and are often associated with aberrant expression or localization of connexins. Aberrant localization of connexins in some tumour cells is associated with lack of function of cell adhesion molecules, suggesting the importance of cell-cell recognition for GJIC. Transfection of connexin genes into tumorigenic cells restores normal cell growth, supporting the idea that connexins form a family of tumour-suppressor genes. Some studies also show that specific connexins may be necessary to control growth of specific cell types. We have produced various dominant-negative mutants of Cx26, Cx32 and Cx43 and showed that some of them prevent the growth control exerted by the corresponding wild-type genes. However, we have found that connexins 32, 37 and 43 genes are rarely mutated in tumours. In some of these studies, we noted that connexin expression per se, rather than GJIC level, is more closely related to growth control, suggesting that connexins may have a GJIC-independent function. We have recently created a transgenic mouse strain in which a mutant Cx32 is specifically overexpressed in the liver. Studies with such mice indicate that Cx32 plays a key role in liver regeneration after partial hepatectomy. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through GJIC. Recently, we and others have shown that GJIC is responsible for the bystander effect seen in HSV-tk/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.     

Connexin43 phosphorylation and cytoprotection in the heart

The fundamental role played by connexins including connexin43 (Cx43) in forming intercellular communication channels (gap junctions), ensuring electrical and metabolic coupling between cells, has long been recognized and extensively investigated. There is also increasing recognition that Cx43, and other connexins, have additional roles, such as the ability to regulate cell proliferation, migration, and cytoprotection. Multiple phosphorylation sites, targets of different signaling pathways, are present at the regulatory, C-terminal domain of Cx43, and contribute to constitutive as well as transient phosphorylation Cx43 patterns, responding to ever-changing environmental stimuli and corresponding cellular needs. The present paper will focus on Cx43 in the heart, and provide an overview of the emerging recognition of a relationship between Cx43, its phosphorylation pattern, and development of resistance to injury. We will also review our recent work regarding the role of an enhanced phosphorylation state of Cx43 in cardioprotection. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Channel-independent influence of connexin 43 on cell migration

In this review we focus on the role of connexins, especially of Cx43, as modulators of migration - a fundamental process in embryogenesis and in physiologic functions of the adult organism. This impact of connexins is partly mediated by their function as intercellular channels but an increasing number of studies support the view that at least part of the effects are truly independent of the channel function. The channel-independent function comprises extrinsic guidance of migrating cells due to connexin mediated cell adhesion as well as intracellular processes. Cx43 has been shown to exert effects on migration by interfering with receptor signalling, cytoskeletal remodelling and tubulin dynamics. These effects are mainly dependent on the presence of the carboxyl tail of Cx43. The molecular basis of this channel-independent connexin function is still not yet fully understood but early results open an exciting view towards new functions of connexins in the cell. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin.     

Intracellular trafficking pathways in the assembly of connexins into gap junctions

Trafficking pathways underlying the assembly of connexins into gap junctions were examined using living COS-7 cells expressing a range of connexin-aequorin (Cx-Aeq) chimeras. By measuring the chemiluminescence of the aequorin fusion partner, the translocation of oligomerized connexins from intracellular stores to the plasma membrane was shown to occur at different rates that depended on the connexin isoform. Treatment of COS-7 cells expressing Cx32-Aeq and Cx43-Aeq with brefeldin A inhibited the movement of these chimera to the plasma membrane by 84 +/- 4 and 88 +/- 4%, respectively. Nocodazole treatment of the cells expressing Cx32-Aeq and Cx43-Aeq produced 29 +/- 16 and 4 +/- 7% inhibition, respectively. In contrast, the transport of Cx26 to the plasma membrane, studied using a construct (Cx26/43T-Aeq) in which the short cytoplasmic carboxyl-terminal tail of Cx26 was replaced with the extended carboxyl terminus of Cx43, was inhibited 89 +/- 5% by nocodazole and was minimally affected by exposure of cells to brefeldin A (17 +/-11%). The transfer of Lucifer yellow across gap junctions between cells expressing wild-type Cx32, Cx43, and the corresponding Cx32-Aeq and Cx43-Aeq chimeras was reduced by nocodazole treatment and abolished by brefeldin A treatment. However, the extent of dye coupling between cells expressing wild-type Cx26 or the Cx26/43T-Aeq chimeras was not significantly affected by brefeldin A treatment, but after nocodazole treatment, transfer of dye to neighboring cells was greatly reduced. These contrasting effects of brefeldin A and nocodazole on the trafficking properties and intercellular dye transfer are interpreted to suggest that two pathways contribute to the routing of connexins to the gap junction.