Interplay between PKC and the MAP kinase pathway in Connexin43 phosphorylation and inhibition of gap junction intercellular communication

Gap junction channels are made of a family proteins called connexins. The best-studied type of connexin, Connexin43 (Cx43), is phosphorylated at several sites in its C-terminus. The tumor-promoting phorbol ester TPA strongly inhibits Cx43 gap junction channels. In this study we have investigated mechanisms involved in TPA-induced phosphorylation of Cx43 and inhibition of gap junction channels. The data show that TPA-induced inhibition of gap junction intercellular communication (GJIC) is dependent on both PKC and the MAP kinase pathway. The data suggest that PKC-induced activation of MAP kinase partly involves Src-independent trans-activation of the EGF receptor, and that TPA-induced shift in SDS-PAGE gel mobility of Cx43 is caused by MAP kinase phosphorylation, whereas phosphorylation of S368 by PKC does not alter gel migration of Cx43. We also show that TPA, in addition to phosphorylation of S368, also induces phosphorylation of S255 and S262, in a MAP kinase-dependent manner. The data add to our understanding of the molecular mechanisms involved in the interplay between signaling pathways in regulation of GJIC.     

ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

We have previously reported that protein kinase C gamma (PKC-gamma) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKC-gamma translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKC-gamma-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKC-gamma with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKC-gamma, the Cx43 did not disassemble out of plaques even though the PKC-gamma enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKC-gamma-driven disassembly response.     

Effect of thioridazine on gap junction intercellular communication in connexin 43-expressing cells

Propagation of electrical activity between myocytes in the heart requires gap junction channels, which contribute to coordinated conduction of the heartbeat. Some antipsychotic drugs, such as thioridazine and its active metabolite, mesoridazine, have known cardiac conduction side-effects, which have resulted in fatal or nearly fatal clinical consequences in patients. The physiological mechanisms responsible for these cardiac side-effects are unknown. We tested the effect of thioridazine and mesoridazine on gap junction-mediated intercellular communication between cells that express the major cardiac gap junction subtype connexin 43. Micromolar concentrations of thioridazine and mesoridazine inhibited gap junction-mediated intercellular communication between WB-F344 epithelial cells in a dose-dependent manner, as measured by fluorescent dye transfer. Kinetic analyses demonstrated that inhibition by 10 μmol/L thioridazine occurred within 5 min, achieved its maximal effect within 1 h, and was maintained for at least 24 h. Inhibition was reversible within 1 h upon removal of the drug. Western blot analysis of connexin 43 in a membrane-enriched fraction of WB-F344 cells treated with thioridazine revealed decreased amounts of unphosphorylated connexin 43, and appearance of a phosphorylated connexin 43 band that co-migrated with a “hyperphosphorylated” connexin 43 band present in TPA-inhibited cells. When tested for its effects on cardiomyocytes isolated from neonatal rats, thioridazine decreased fluorescent dye transfer between colonies of beating myocytes. Microinjection of individual cells with fluorescent dye also showed inhibition of dye transfer in thioridazine-treated cells compared to vehicle-treated cells. In addition, thioridazine, like TPA, inhibited rhythmic beating of myocytes within 15 min of application. In light of the fact that the thioridazine and mesoridazine concentrations used in these experiments are in the range of those used clinically in patients, our results suggest that inhibition of gap junction intercellular communication may be one factor contributing to the cardiac side-effects observed in some patients taking these medications.     

An activator of protein kinase C inhibits gap junction communication between cultured bovine lens cells.

Currently little is known about the regulation of gap junction communication in the lens. We report here on the effects of the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on cultured bovine lens cells which appeared to be epithelial in nature. Dramatically reduced intercellular transfer of the fluorescent dye Lucifer yellow was observed when the cultured lens cells were treated with octanol, a known inhibitor of gap junction communication. TPA (4 beta isomer) was also shown to reduce intercellular permeability within these cultures. In contrast, an inactive form of TPA, 4 alpha-TPA, did not decrease dye transfer. Permeability was evaluated in terms of both the number of cells receiving dye and the rate of decrease in fluorescence intensity in the injected cell. The maximum decreases in dye transfer occurred at 2 h of TPA treatment and dye transfer gradually increased to control levels over a time course of many hours. Incubation of cultures with 32Pi and immunoprecipitation using antibodies to the N- and C-terminal regions of connexin43 demonstrated a gap junction phosphoprotein of 43,000 Da. Phosphorylation of connexin43 increased during the first 2 h of TPA treatment. These results suggest that protein kinase C has a direct or indirect effect on gap junction communication in cultured lens cells.     

Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.     

Connexin43 in porcine myocardium and non-pregnant myometrium

It is generally accepted that connexin43 (Cx43) is a major constituent of heart and myometrial gap junctions. However, the presence of Cx43 gap junctions in non-pregnant myometrium is still poorly documented. Tissue sections of porcine heart and non-pregnant uterus and myometrial smooth muscle cell cultures were immunostained with monoclonal antibody against Cx43. In the heart, intensive immunostaining was confined to the intercalated discs as previously reported. In the non-pregnant uterus, punctuate immunostaining of Cx43 was seen throughout the myometrium along cell interfaces between myocytes. The expression of Cx43 was sustained in cultured smooth muscle cells isolated from non-pregnant myometrium. Western blotting has detected single isoform of Cx43 in both, cardiac and myometrial tissues. The electrophoretic mobility of porcine heart Cx43 was similar to that of myometrial isoform but different from the pattern of mobility of Cx43 of the rat heart. Hence, porcine myometrium may provide attractive model for studying cellular mechanisms triggering expression of gap junction protein in normal (non-pregnant) uterus.     

Connexin43 in MDCK cells: regulation by a tumor-promoting phorbol ester and Ca2+.

Prior to confluence, cultures of Madin Darby canine kidney (MDCK) cells expressed gap junctional communication, as assessed by fluorescent dye transfer, as well as relatively high levels of an anti-connexin43 immunoreactive component referred to as connexin43 (Cx43). After confluence, dye coupling and levels of Cx43 were dramatically reduced. Immunofluorescence analysis of the distribution of Cx43 in subconfluent cultures showed punctate labeling on the plasma membrane at regions of cell apposition and a more diffuse labeling in perinuclear regions. Western blots of total cell homogenates showed that the dephosphorylated form of Cx43 was more abundant than the phosphorylated forms. Phosphorylation of Cx43 was not significantly affected by 8-Bromo-cAMP or 8-Bromo-cGMP. However, 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited dye coupling and induced an increase in the amount of phosphorylated forms of Cx43 at the expense of the dephosphorylated form. This effect occurred as rapidly as 5 min after TPA treatment without apparent changes in distribution of Cx43 or cell morphology. These results suggest that second messenger pathways involving protein kinase C, but not cAMP- or cGMP-dependent protein kinase, led to changes in electrophoretic mobility of Cx43, revealed by Western blot, consistent with an alteration in the state of phosphorylation of the gap junction protein. Treatments with staurosporine, a protein kinase inhibitor, or okadaic acid, a protein phosphatase inhibitor, either alone or in combination with TPA, indicated that the abundance of the dephosphorylated form of Cx43 in MDCK cells was due to low kinase activity. It was also found that lowering the concentration of extracellular Ca2+, which reduced cell contact, did not affect the abundance, the state of phosphorylation, or the TPA-induced phosphorylation of Cx43. These results suggest that neither extracellular Ca2+ nor cell contact is required for basal or TPA-induced phosphorylation of Cx43.     

Cultured myometrial cells establish communicating gap junctions

Myometrial cells were isolated and cultured from term rat uterus. The myometrial origin of the cultures was verified by antibody staining of cellular desmin and alpha-smooth muscle actin. The presence of functional gap junctions was indicated by transfer of radiolabeled nucleotide and microinjected Lucifer yellow dye. The cultured cells expressed mRNA recognized by a connexin43 gap junction cDNA probe. To our knowledge, this is the first report that isolated myometrial cells form gap junctions in culture.     

Phosphatases Involved in Modulation of Gap Junctional Intercellular Communication and Dephosphorylation of Connexin43 in Hamster Fibroblasts: 2B or Not 2B?

12-O-Tetradecanoylphorbol-13-acetate (TPA) caused strong suppression of gap junctional intercellular communication, altered phosphorylation status of the gap junction protein, connexin43, and disappearance of immunorecognizible connexin43-containing gap junction plaques in V79 fibroblasts. When TPA was removed, all parameters normalized during a 3- to 4-h period. The normalizations were independent of protein synthesis, suggesting the possible involvement of phosphatases. None of the phosphatase inhibitors okadaic acid, calyculin A, cyclosporin A, or FK506 affected intercellular communication or connexin43 phosphorylation status on their own. In sequential exposures to TPA and phosphatase inhibitors, only the protein-phosphatase 2B (PP2B) inhibitors cyclosporin A and FK506 delayed the recovery of the studied parameters. Rapamycin binds to the same set of proteins as does FK506, but without inhibiting PP2B. Rapamycin did not affect the recovery of intercellular communication, but it delayed the normalization of connexin43 band pattern and immunorecognition of gap junction plaques. Dephosphorylation of immunoprecipitated connexin43 was studied using PP1, 2A, 2B, and 2C. PP2A was the most efficient (by 100-fold on a molar basis). Connexin43 immunoprecipitated from TPA-exposed cells was a poor substrate for PP1, 2B, and 2C. Thus, PP2B appeared to play a role in normalization of intercellular communication, but not necessarily in direct dephosphorylation of connexin43. Peptidyl-prolyl isomerase activity of cyclosporin/FK506/rapamycin-binding proteins may promote the dephosphorylation of connexin43 in cells.     

2,2'-Dichlorobiphenyl decreases amplitude and synchronization of uterine contractions through MAPK1-mediated phosphorylation of GJA1 (connexin43) and inhibition of myometrial gap junctions

The present study examined the hypothesis that inhibition of myometrial gap junctions through MAPK1-induced phosphorylation of GJA1 (connexin43) leads to inhibition of spontaneous phasic uterine contractions by 2,2'-dichlorobiphenyl (2,2'-DCB). Uterine strips from Gestation Day 10-pregnant rats exposed in muscle baths to 2,2'-DCB exhibited increased oscillatory frequency and decreased amplitude and synchronization of contractions. To assess effects on gap junctions, Lucifer yellow was injected into myometrial cells and transfer to adjacent cells was scored. After a 1-h treatment, 100 microM 2,2'-DCB decreased Lucifer yellow intercellular transfer in a concentration-dependent manner. The MAP2K1 inhibitor PD98059 increased percentage of dye transfer to adjacent myometrial cells from 18% in cultures exposed for 1 h to 100 microM 2,2'-DCB alone to 48% in cultures cotreated with 50 microM PD98059 and 100 microM 2,2'-DCB. In contrast, the conventional PRKC inhibitor G?6976 (10 microM) had no significant effect on 2,2'-DCB-induced inhibition of dye transfer. Western blotting showed about a 4.5-fold increase in phosphorylation of GJA1 at S255, a MAPK1 site, after exposure to 100 microM 2,2'-DCB compared to untreated and solvent controls. However, there was no difference in phosphorylation of GJA1 at S368, a PRKC site. Cells treated with 2,2'-DCB increased phosphorylated MAPK1, implicating the increase of activation of MAPK1. Cotreatment with 100 microM 2,2'-DCB and 5 microM PD98059 reversed 2,2'-DCB-induced modification of uterine contractions and increase of pGJA1(S255) in uterine strips. Therefore, this study suggests that 2,2'-DCB decreases amplitude and synchronization of uterine contractions mediated through MAPK1-mediated phosphorylation of GJA1 and subsequent inhibition of myometrial gap junctions.     

Protein kinase C spatially and temporally regulates gap junctional communication during human wound repair via phosphorylation of connexin43 on serine368

Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance. Examination of phosphoserine368 (pS368) in normal human skin tissue using a phosphorylation site-specific antibody showed relatively even distribution throughout the epidermal layers. However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 microm of it). Scratch wounding of primary human keratinocytes caused a protein kinase C (PKC)-dependent increase in pS368 in cells adjacent to the scratch, with a time course similar to that found in the wounds. Keratinocytes at the edge of the scratch also transferred dye much less efficiently at 24 h, in a manner dependent on PKC. However, keratinocyte migration to fill the scratch required early (within <6 h) gap junctional communication. Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.     

Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.     

Inhibition of connexin43 gap junctional intercellular communication by TPA requires ERK activation

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent inhibitor of gap junctional intercellular communication (GJIC). This inhibition requires activation of protein kinase C (PKC), but the events downstream of this kinase are not known. Since PKC can activate extracellular signal regulated kinases (ERKs) and these also downregulate GJIC, we hypothesized that the inhibition of GJIC by TPA involved ERKs. TPA treatment (10 ng/ml for 30 min) of WB-F344 rat liver epithelial cells strongly activated p42 and p44 ERK-1 and -2, blocked gap junction-mediated fluorescent dye-coupling, and induced connexin43 hyperphosphorylation and gap junction internalization. These effects were completely prevented by inhibitors of PKC (bis-indolylmaleimide I; 2 microM) and ERK activation (U-0126; 10 microM). These data suggest that ERKs are activated by PKC in response to TPA treatment and are downstream mediators of the gap junction effects of the phorbol ester.     

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.     

Changes in Connexin43 Expression and Localization During Pancreatic Cancer Progression

Gap junctions and gap junction communication have long been recognized to play roles in tissue organization and remodeling through both cell autonomous and intercellular means. We hypothesized that these processes become dysregulated during pancreas cancer progression. Molecular and histological characterization of the gap junction protein, connexin43, during progression of pancreatic ductal adenocarcinoma could yield insight into how these events may contribute to or be modulated during carcinogenesis. In a mouse model of pancreatic ductal adenocarcinoma generated through targeted endogenous expression of Kras(G12D) in the murine pancreas, we examined the evolving expression and localization of connexin43. Overall, connexin43 expression increased over time, and its localization became more widespread. At early stages, connexin43 is found almost exclusively in association with the basolateral membrane of duct cells found in invasive lesions. Connexin43 became increasingly associated with the surrounding stroma over time. Connexin43 phosphorylation was also altered during tumorigenesis, as assessed by migrational changes of the protein in immunoblots. These data suggest a potential role for gap junctions and connexin43 in mediating interactions between and amongst the stromal and epithelial cells in pancreatic ductal adenocarcinoma.     

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

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

Degradation of Connexin43 Gap Junctions Involves both the Proteasome and the Lysosome

Intercellular communication may be modulated by the rather rapid turnover and degradation of gap junction proteins, since many connexins have half-lives of 1–3 h. While several morphological studies have suggested that gap junction degradation occurs after endocytosis, our recent biochemical studies have demonstrated involvement of the ubiquitin–proteasome pathway in proteolysis of the connexin43 polypeptide. The present study was designed to reconcile these observations by examining the degradation of connexin43-containing gap junctions in rat heart-derived BWEM cells. After treatment of BWEM cells with Brefeldin A to prevent transport of newly synthesized connexin43 polypeptides to the plasma membrane, quantitative confocal microscopy showed the disappearance of immunoreactive connexin43 from the cell surface with a half-life of 1 h. This loss of connexin43 immunoreactivity was inhibited by cotreatment with proteasomal inhibitors (ALLN, MG132, or lactacystin) or lysosomal inhibitors (leupeptin or E-64). Similar results were seen when connexin43 export was blocked with monensin. After treatment of BWEM cells with either proteasomal or lysosomal inhibitors alone, immunoblots showed accumulation of connexin43 in both whole cell lysates and in a 1% Triton X-100-insoluble fraction. Immunofluorescence studies showed that connexin43 accumulated at the cell surface in lactacystin-treated cells, but in vesicles in BWEM cells treated with lysosomal inhibitors. These results implicate both the proteasome and the lysosome in the degradation of connexin43-containing gap junctions.     

Identification and functional analysis of novel phosphorylation sites in Cx43 in rat primary granulosa cells

The gap junctional intercellular communication mediated by Cx43 plays indispensable roles in both germ line development and postnatal folliculogenesis. In this study, we focused on the effect of follicle-stimulating hormone (FSH) on the Cx43 protein in rat primary granulosa cells and found that FSH stimulation elevated the phosphorylation in addition to the protein level of Cx43. Serine residues in the carboxyl-terminal region were exclusively phosphorylated in this system and we identified Ser365, Ser368, Ser369 and Ser373 as major phosphorylation sites by FSH stimulation. A Cx43 variant containing mutations at all these serine residues was found to severely reduce dye transfer activity when assayed in HeLa cells. The present study revealed a novel regulatory mechanism of Cx43-mediated gap junctional intercellular communication through phosphorylation in the carboxyl-terminus.     

Ilimaquinone inhibits gap junctional communication in a connexin isotype-specific manner

Ilimaquinone (IQ) and brefeldin A (BFA) disrupt the Golgi complex structure and block protein transport to the plasma membrane, and inhibit gap junctional communication. HeLa cells expressing rat connexin26, 32, or 43, or mouse connexin31, 36, 45, or 57, were used to study the response patterns of gap junctional communication (dye transfer) to ilimaquinone, brefeldin, and the potent protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA). 12-O-Tetradecanoylphorbol-13-acetate (followed for 2 h) caused dose- and time-dependent decreases in communication for five of seven connexins, the unresponsive being connexin45 and 57. Brefeldin (followed for 6 h) caused dose- and time-dependent decreases in communication for six of seven connexins, the exception being connexin26. These results are consistent with Golgi-mediated transport to the cell membrane for all connexins except connexin26. In contrast, ilimaquinone (followed for 6 h) caused a rapid (15-30 min) and nearly complete inhibition of dye transfer through connexin43 channels. For the other connexins, there was a slow and weak response for connexin26, 31, and 32, reaching 65-70% of control communication level, while connexin36, 45, and 57 were unresponsive. Thus, among the tested connexins, ilimaquinone has a strong specificity for connexin43, and the mechanism appears independent of the Golgi complex and of protein kinase C.