Different domains are critical for oligomerization compatibility of different connexins

Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies.     

Intracellular Domains of Mouse Connexin26 and -30 Affect Diffusional and Electrical Properties of Gap Junction Channels

To evaluate the influence of intracellular domains of connexin (Cx) on channel transfer properties, we analyzed mouse connexin (Cx) Cx26 and Cx30, which show the most similar amino acid sequence identities within the family of gap junction proteins. These connexin genes are tightly linked on mouse chromosome 14. Functional studies were performed on transfected HeLa cells stably expressing both mouse connexins. When we examined homotypic intercellular transfer of microinjected neurobiotin and Lucifer yellow, we found that gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer yellow. Furthermore, we observed heterotypic transfer of neurobiotin between Cx30-transfectants and HeLa cells expressing mouse Cx30.3, Cx40, Cx43 or Cx45, but not between Cx26 transfectants and HeLa cells of the latter group. The main differences in amino acid sequence between Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-terminal region of these integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30 chimeric cDNAs, which were also expressed in HeLa cells after transfection. Homotypic intercellular transfer of injected Lucifer yellow was observed exclusively with those chimeric constructs that coded for both cytoplasmic domains of Cx26 in the Cx30 backbone polypeptide chain. In contrast, cells transfected with a construct that coded for the Cx26 backbone with the Cx30 cytoplasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these connexins. In turn, the cytoplasmic loop and C-terminal domain of Cx30 prevent Lucifer yellow transfer when swapped with the corresponding domains of Cx26. In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal domains had been exchanged, the unitary channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly reduced. This suggests that these cytoplasmic domains interfere directly or indirectly with the diffusivity, the conductance and voltage gating of the channels. Received: 26 July 2000/Revised: 15 February 2001     

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.     

Inducible Coexpression of Connexin37 or Connexin40 with Connexin43 Selectively Affects Intercellular Molecular Transfer

Many tissues express multiple gap junction proteins, or connexins (Cx); for example, Cx43, Cx40, and Cx37 are coexpressed in vascular cells. This study was undertaken to elucidate the consequences of coexpression of Cx40 or Cx37 with Cx43 at different ratios. EcR-293 cells (which endogenously produce Cx43) were transfected with ecdysone-inducible plasmids encoding Cx37 or Cx40. Immmunoblotting showed a ponasterone dose-dependent induction of Cx37 or Cx40 while constant levels of Cx43 were maintained. The coexpressed connexins colocalized at appositional membranes. Double whole-cell patch clamp recordings showed no significant change in total junctional conductances in cells treated with 0, 0.5, or 4?μM ponasterone; however, they did show a diversity of unitary channel sizes consistent with the induced connexin expression. In cells with induced expression of either Cx40 or Cx37, intercellular transfer of microinjected Lucifer yellow was reduced, but transfer of NBD-TMA (2-(4-nitro-2,1,3-benzoxadiol-7-yl)[aminoethyl]trimethylammonium) was not affected. In cocultures containing uninduced EcR cells together with cells induced to coexpress Cx37 or Cx40, Lucifer yellow transfer was observed only between the cells expressing Cx43 alone. These data show that induced expression of either Cx37 or Cx40 in Cx43-expressing cells can selectively alter the intercellular exchange of some molecules without affecting the transfer of others.     

A Carboxyl Terminal Domain of Connexin43 Is Critical for Gap Junction Plaque Formation but not for Homo- or Hetero-Oligomerization

We have initiated a series of experiments to analyze the biosynthesis and oligomerization of Cx43 in cells containing other connexins through the expression of site-directed mutants and chimeric connexin polypeptides. Here we report studies concerning a mutant of Cx43 (Cx43tr) that has been truncated after amino acid 251 to remove most of the Cx43 carboxy-terminal region. In stably transfected HeLa cells, full length Cx43 localized primarily to appositional membranes while much more Cx43tr was observed in the cytoplasm. Both Cx43 and Cx43tr showed similar oligomerization profiles based on centrifugation through sucrose gradients. HeLaCx43tr cells showed limited transfer of microinjected Lucifer Yellow but did show electrical coupling. Co-expression of Cx43tr with Cx43 or Cx45 led to Cx43tr localization at appositional membranes and co-localization with the other connexins. Moreover, cells co-expressing Cx43tr with Cx43 or Cx45 showed extensive intercellular dye coupling. Thus, Cx43tr was able to oligomerize and form functional channels when expressed alone or with a compatible connexin, but it only formed plaques when co-expressed. These results suggest that the carboxyl tail of Cx43 is not important for oligomerization, but they implicate critical residues in the formation of gap junction plaques.     

A carboxyl terminal domain of connexin43 is critical for gap junction plaque formation but not for homo- or hetero-oligomerization

We have initiated a series of experiments to analyze the biosynthesis and oligomerization of Cx43 in cells containing other connexins through the expression of site-directed mutants and chimeric connexin polypeptides. Here we report studies concerning a mutant of Cx43 (Cx43tr) that has been truncated after amino acid 251 to remove most of the Cx43 carboxy-terminal region. In stably transfected HeLa cells, full length Cx43 localized primarily to appositional membranes while much more Cx43tr was observed in the cytoplasm. Both Cx43 and Cx43tr showed similar oligomerization profiles based on centrifugation through sucrose gradients. HeLaCx43tr cells showed limited transfer of microinjected Lucifer Yellow but did show electrical coupling. Co-expression of Cx43tr with Cx43 or Cx45 led to Cx43tr localization at appositional membranes and co-localization with the other connexins. Moreover, cells co-expressing Cx43tr with Cx43 or Cx45 showed extensive intercellular dye coupling. Thus, Cx43tr was able to oligomerize and form functional channels when expressed alone or with a compatible connexin, but it only formed plaques when co-expressed. These results suggest that the carboxyl tail of Cx43 is not important for oligomerization, but they implicate critical residues in the formation of gap junction plaques.     

Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery

Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26, 43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 microM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 microM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor 40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.     

Overexpression of cardiac connexin45 increases susceptibility to ventricular tachyarrhythmias in vivo

Electrophysiological remodeling involving gap junctions has been demonstrated in failing hearts and may contribute to intercellular uncoupling, delayed conduction, enhanced arrhythmias, and vulnerability to sudden death in patients with heart failure. Recently, we showed that failing human hearts exhibit marked increases in connexin45 (Cx45) expression in addition to previously documented decreases in connexin43 (Cx43) expression. Each of these changes results in reduced gap junction coupling. The objective of the present study was to examine functional consequences of increased Cx45 in cardiac gap junctions. Transgenic mice with cardiac-selective overexpression of the developmentally downregulated cardiac connexin, connexin45 (Cx45OE mice) were subjected to in vivo electrophysiology studies in which an intracardiac catheter was used to induce ventricular arrhythmias in anesthetized mice, and in which ambulatory ECG monitoring was used to detect spontaneous arrhythmias in unanesthetized mice. Hearts were analyzed by TaqMan RT-PCR, immunostaining, immunoblotting, and echocardiography. Lucifer yellow and neurobiotin dye transfer was used to assess coupling in transgenic and control myocyte cultures. Cx45 mRNA was two orders of magnitude greater in Cx45OE mice. Cx45-immunoreactive signal at gap junctions increased twofold and total Cx45 protein by immunoblotting increased 25% in Cx45OE mice compared with nontransgenic littermate controls. Functionally, Cx45OE mice exhibited more inducible ventricular tachycardia than controls but did not exhibit any other functional or structural derangements as assessed by echocardiography. Ventricular myocytes isolated from Cx45OE mice exhibited diminished intercellular transfer of Lucifer yellow dye and increased transfer of neurobiotin, consistent with altered cell-to-cell communication. Thus increased myocardial expression of Cx45 results in remodeling of intercellular coupling and greater susceptibility to ventricular arrhythmias in vivo.     

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha- type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.     

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.     

Gap junction permeability: selectivity for anionic and cationic probes

Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the permeability of homotypic connexin26 (Cx26), Cx40, Cx43, and Cx45 gap junction channels stably expressed in HeLa cells to solutes with different size and net charge. Channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe. All four connexins allowed passage of both cationic and anionic probes, but the transfer rates were connexin dependent. The negatively charged probes [Lucifer yellow (LY; median axial diameter 9.9 ?, charge -2), carboxyfluorescein (CF; 8.2 ?; -2), and Alexa Fluor350 (AF350, 5.4 ?; -1)] exhibited the following permeability order: Cx43 > Cx45 > Cx26 > Cx40. In contrast, for the positively charged species permeability, the orders were as follows: Cx26 ≈ Cx43 ≈ Cx40 ≈ Cx45 for N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl) amino] ethanaminium (NBD-m-TMA; 5.5 ?, +1) and Cx26 ≥ Cx43 ≈ Cx40 > Cx45 for ethidium bromide (10.3 ?, +1). Comparison of probe permeability relative to K(+) revealed that Cx43 and Cx45 exhibited similar permeability for NBD-m-TMA and AF350, indicating weak charge selectivity. However, lesser transfer of CF and LY through Cx45 relative to Cx43 channels suggests stronger size-dependent discrimination of solute. The permeability of NBD-m-TMA for Cx40 and Cx26 channels was approximately three times higher than to anionic AF350 despite the fact that both have similar minor diameters, suggesting charge selectivity. In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling.     

Wounding alters epidermal connexin expression and gap junction-mediated intercellular communication.

We show that connexin expression and in vivo patterns of communication were dramatically altered in response to epidermal wounding. Six hours after injury, Cx26 was up-regulated in the differentiated cells proximal to the wound, but was down-regulated in cells located at the wound edge. In contrast, Cx31.1 and Cx43 were down-regulated in cells both peripheral to and at the wounded edge. These patterns of altered connexin expression were detectable as early as 2 h after wounding and were most pronounced in 24-h old wounds. Increased expression of Cx26 was still evident in the hyperproliferative epidermis of 6-day old wounds. In vivo dye transfer experiments with Lucifer yellow and neurobiotin confirmed that junctional communication patterns were altered in ways consistent with changes in connexin expression. The data thus suggest that intercellular communication is intimately involved in regulating epidermal wound repair.     

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.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Gap junction assembly: multiple connexin fluorophores identify complex trafficking pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45

We examined the permeabilities of homotypic and heterotypic gap junction (GJ) channels formed of rodent connexins (Cx) 30.2, 40, 43, and 45, which are expressed in the heart and other tissues, using fluorescent dyes differing in net charge and molecular mass. Combining fluorescent imaging and electrophysiological recordings in the same cell pairs, we evaluated the single-channel permeability (P(gamma)). All homotypic channels were permeable to the anionic monovalent dye Alexa Fluor-350 (AF(350)), but mCx30.2 channels exhibited a significantly lower P(gamma) than the others. The anionic divalent dye Lucifer yellow (LY) remained permeant in Cx40, Cx43, and Cx45 channels, but transfer through mCx30.2 channels was not detected. Heterotypic channels generally exhibited P(gamma) values that were intermediate to the corresponding homotypic channels. P(gamma) values of mCx30.2/Cx40, mCx30.2/Cx43, or mCx30.2/Cx45 heterotypic channels for AF(350) were similar and approximately twofold higher than P(gamma) values of mCx30.2 homotypic channels. Permeabilities for cationic dyes were assessed only qualitatively because of their binding to nucleic acids. All homotypic and heterotypic channel configurations were permeable to ethidium bromide and 4,6-diamidino-2-phenylindole. Permeability for propidium iodide was limited only for GJ channels that contain at least one mCx30.2 hemichannel. In summary, we have demonstrated that Cx40, Cx43, and Cx45 are permeant to all examined cationic and anionic dyes, whereas mCx30.2 demonstrates permeation restrictions for molecules with molecular mass over approximately 400 Da. The ratio of single-channel conductance to permeability for AF(350) was approximately 40- to 170-fold higher for mCx30.2 than for Cx40, Cx43, and Cx45, suggesting that mCx30.2 GJs are notably more adapted to perform electrical rather than metabolic cell-cell communication.     

Phosphorylated Carboxy Terminal Serine Residues Stabilize the Mouse Gap Junction Protein Connexin45 Against Degradation

Phosphoamino acid analysis of mouse connexin45 (Cx45) expressed in human HeLa cells revealed that phosphorylation occurred mainly at serine residues, but also on tyrosine and threonine residues. To characterize the role of Cx45 phosphorylation, different serine residues of the serine-rich carboxy terminal region were deleted or exchanged for other amino acids residues. Human HeLa cells deficient in gap junctional intercellular communication were stably transfected with appropriate constructs and analyzed for expression, localization, phosphorylation, formation of functional gap junction channels and degradation of mutant Cx45. After exchange or deletion of nine carboxy terminal serine residues, phosphorylation was decreased by 90%, indicating that these serine residues represented main phosphorylation sites of mouse Cx45. The various serine residues of this region contributed differently to the phosphorylation of Cx45 suggesting a cooperative mechanism for phosphorylation. Substitution of different serine residues for other amino acids did not interfere with correct intracellular trafficking and assembly of functional gap junction channels, as shown by localization of mutant Cx45 at the plasma membrane and by dye transfer to neighboring cells. Truncated Cx45 was also weakly phosphorylated but was trapped in perinuclear locations. Dye transfer of these transfectants was similar as in nontransfected HeLa cells. The half-life of mouse Cx45 protein in HeLa cells was determined as 4.2 hr. Pulse-chase experiments with the different transfectants revealed an increased turnover of Cx45, when one or both of the serine residues at positions 381 and 382 or 384 and 385 were exchanged for other amino acids. The half-life of these mutants was diminished by 50% compared to wild type Cx45. Received: 26 September 1997/Revised: 5 January 1997     

Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes

In the present study, we have analyzed the direct effects of cytokines, which mediate the acute-phase response in liver, on connexin expression and gap-junctional intercellular communication in immortalized MHSV12 mouse hepatocytes. When these cells were stimulated for 24 h with interleukin 1 and interleukin 6, the amount of connexin26 (Cx26) mRNA increased together with β?fibrinogen mRNA, as expected for this positive acute-phase gene. In contrast, connexin32 (Cx32) mRNA expression was not affected under these conditions. Indirect immunfluorescence revealed a drastic decrease in Cx32 signals, whereas slightly more Cx26 signals were found. Stronger stimulation with interleukin 1 and tumor necrosis factor α gave a dose-dependent increase in steady state levels of Cx26 and β-fibrinogen mRNA, but no further change in Cx32 mRNA level was seen. However, when Cx32 protein was analyzed on immunoblots, we found a 5-fold decrease in expression even at low cytokine doses that did not affect Cx32 mRNA expression. Under these conditions, cell to cell transfer of Lucifer yellow, microinjected into immortalized hepatocytes, was decreased by 70%, suggesting that intercellular communication through Cx32 channels was partially inhibited earlier than other genetic alterations characteristic of the acute-phase response. Thus, the major hepatic gap junction protein was largely downregulated at the beginning of the experimental inflammatory reaction, but about 30% of gap-junctional intercellular communication was maintained. This suggests that, during the acute-phase response, the second hepatic Cx26 protein may compensate in part for the downregulation of the Cx32 protein.