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

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

Length of C-terminus of rCx46 influences oligomerization and hemichannel properties

Wild type connexin 46 of rat (wtrCx46), and human connexin 26 (wthCx26) and derivates from rCx46 elongated at the C-terminus by 25 amino acids (rCx46Ct) as well as C-terminal truncated constructs (rCx28.1, rCx45.3) were expressed in frog oocytes of Xenopus laevis. Single oocyte voltage-clamp analysis revealed that connexons or hemichannels of rCx46Ct exhibit similar conducting properties as those of wtrCx46. Insertion of a stop codon at C-terminal domains at position 243 and 409 resulted in a significant reduction in the corresponding hemichannel conductance. This result was also found for wthCx26, the shortest human connexin. Tagged connexin constructs rCx46Ct and hCx26Ct could be expressed in E. coli as monomers. The monomers of rCx46Ct and hCx26Ct were purified and electro-eluted from corresponding SDS gels. Studies of in vitro oligomerization showed that hexamers of these connexins were formed in presence of kinase and specific lipids. Purified rCx46Ct formed some oligomers in vitro if a lipid mixture of POPE/POPG and casein kinase I (CKI) was added, but in the presence of POPC, phosphorylated rCx46Ct monomers preferentially formed hexamers. Purified hCx26Ct formed hexamers in the presence of POPE/POPG. In addition, N-terminal truncated rCx46 (Cx35) oligomerized after phosphorylation. Reconstitution of purified recombinant connexin rCx46Ct in planar lipid bilayers mediated Ca(2+)-sensitive single channel activity. It is discussed whether the specific C-terminal end of the expressed connexins are responsible for hexamer formation as well as channel opening.     

Molecular mechanisms regulating formation,trafficking and processing of annular gap junctions

Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena.

     

Relationship of Cytoskeletal Filaments to Annular Gap Junction Expression in Human Adrenal Cortical Tumor Cells in Culture

In addition to the well-characterized surface gap junctions expressed at contact sites between cells, annular gap junction profiles have been localized within the cytoplasm of some cell populations. To study and characterize these annular profiles, gap junction protein type was demonstrated with Western blot and immunocytochemistry. The distribution of annular gap junctions and the relationships to cytoskeletal elements were demonstrated with immunocytochemical, transmission electron microscopic, or image analysis with confocal microscopy techniques. SW-13 adrenal cortical tumor cells expressed α₁gap junctions at areas of cell to cell contact. In addition, α₁gap junction annular profiles were seen within the cytoplasm. Actin and myosin II were found closely associated with these annular gap junctions, while no physical association between tubulin- or vimentin-containing fibers and gap junction protein could be established. Disruption of microfilaments with cytochalasin B treatment (10 μg/ml, 1 h) resulted in a decrease in the average number and an increase in the average size of annular gap junctions compared to control populations. The results are consistent with a role for cytoskeletal elements containing actin and myosin II in annular gap junction turnover.     

Regulation of Lens rCx46-formed Hemichannels by Activation of Protein Kinase C, External Ca2+ and Protons

Rodent lens connexin46 (rCx46) formed active voltage-dependent hemichannels when expressed in Xenopus oocytes. Time-dependent macroscopic currents were evoked upon depolarization. The observed two activation time constants were weakly voltage-dependent and in the order of hundreds of milliseconds and seconds, respectively. Occasionally, the macroscopic steady-state current and the corresponding current-voltage curve showed inactivation at high depolarizing voltages (>+50 mV). To account for the fast recovery from inactivation (<2 msec) favored by hyperpolarization, a four-state kinetic model (C _{1 closed} ↔C _{2 closed} ↔O _open ↔I _inactivated ) is proposed. In the absence of inactivation, the macroscopic conductance decreased and inactivation became visible at voltages positive of +50 mV when the rCx46-expressing oocytes were treated with the protein-kinase-C-activators OAG or TPA, high external concentrations of Ca²⁺ or H⁺. However, the underlying mechanisms of OAG, H⁺ or Ca²⁺ action were different. While OAG did not alter the voltage-dependent activation of the rCx46-hemichannels, an increase in the external Ca²⁺ or H⁺ level shifted the voltage threshold for activation to more positive voltages. In contrast to Ca²⁺, protons were not effective in the physiological concentration range. We propose that under physiological conditions only external Ca²⁺ and intracellular PKC-dependent processes regulate rCx46 in the lens. Received: 30 March 1999/Revised: 18 September 1999     

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.     

Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37

Homomeric gap junction channels are composed solely of oneconnexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different fromeach other. A heteromeric gap junction channel is one that containsdifferent connexins within either or both hemichannels. The existenceof heteromeric forms has been suggested, and many cell types are knownto coexpress connexins. To determine if coexpressed connexins wouldform heteromers, we cotransfected rat connexin43 (rCx43) and humanconnexin37 (hCx37) into a cell line normally devoid of any connexinexpression and used dual whole cell patch clamp to compare the observedgap junction channel activity with that seen in cells transfected onlywith rCx43 or hCx37. We also cocultured cells transfected with hCx37 orrCx43, in which one population was tagged with a fluorescent marker tomonitor heterotypic channel activity. The cotransfected cells possessedchannel types unlike the homotypic forms of rCx43 or hCx37 or theheterotypic forms. In addition, the noninstantaneous transjunctionalconductance-transjunctional voltage(G_j/V_j)relationship for cotransfected cell pairs showed a large range ofvariability that was unlike that of the homotypic or heterotypic form.The heterotypic cell pairs displayed asymmetric voltage dependence. Theresults from the heteromeric cell pairs are inconsistent with summedbehavior of two independent homotypic populations or mixed populationsof homotypic and heterotypic channels types. TheG_j/V_jdata imply that the connexin-to-connexin interactions are significantlyaltered in cotransfected cell pairs relative to the homotypic andheterotypic forms. Heteromeric channels are a population of channelswhose characteristics could well impact differently from theirhomotypic counterparts with regard to multicellular coordinatedresponses.

     

Molecular mechanisms regulating formation,trafficking and processing of annular gap junctions

Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena.     

Immunocytochemical localization of the gap junction 26 K protein in mouse liver plasma membranes

Specific binding sites for anti-26 K antibodies directed against the liver gap junction protein (26 K) were localized by immunoelectron microscopy in gap junction plaques purified from hepatic plasma membranes. Using immunofluorescence microscopy we found discrete fluorescent spots on plasma membranes in cross sections of liver tissues after incubation with anti-26 K antibodies. This is consistent with the notion of specific binding to gap junction plaques. Quantitative binding of anti-26 K antibodies was indirectly measured by the protein A-gold technique. We found that urea/detergent-treated, purified gap junction plaques bind 30-fold more anti-26 K antibodies than preimmune serum. Anti-26 K antibodies also bind specifically to native gap junction plaques within hepatic plasma membranes although only about one fifth as efficiently as to purified plaques. Possibly the anti-26 K antibodies raised after injection of SDS-denatured 26 K protein into rabbits recognize the cytoplasmic face of urea/detergent-treated plaques better than that of native plaques. Some, if not most, of the vesicular structures in preparations of purified plaques appear to be derived from split gap junction plaques and are probably sheets of gap junction hemichannels. In some vesicles the former cytoplasmic face of the hemichannels is turned outside, other vesicles have the former cell surface turned outside. The anti-26 K antibodies do not recognize any 26 K protein on the sheets of partially split gap junction plaques, on the heterogeneous vesicular structures, or on non-junctional areas of hepatic plasma membranes. These results suggest that the conformation of the 26 K protein in plaques must be different from that of the 26 K protein in earlier biosynthetic steps of plaque assembly.     

Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.     

Distribution of Connexin50 Channels and Hemichannels in Lens Fibers: A Structural Approach

A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques.     

Cataract-associated D3Y mutation of human connexin46 (hCx46) increases the dye coupling of gap junction channels and suppresses the voltage sensitivity of hemichannels

Connexin46 (Cx46), together with Cx50, forms gap junction channels between lens fibers and participates in the lens pump-leak system, which is essential for the homeostasis of this avascular organ. Mutations in Cx50 and Cx46 correlate with cataracts, but the functional relationship between the mutations and cataract formation is not always clear. Recently, it was found that a mutation at the third position of hCx46 that substituted an aspartic acid residue with a tyrosine residue (hCx46D3Y) caused an autosomal dominant zonular pulverulent cataract. We expressed EGFP-labeled hCx46wt and hCx46D3Y in HeLa cells and found that the mutation did not affect the formation of gap junction plaques. Dye transfer experiments using Lucifer Yellow (LY) and ethidium bromide (EthBr) showed an increased degree of dye coupling between the cell pairs expressing hCx46D3Y in comparison to the cell pairs expressing hCx46wt. In Xenopus oocytes, two-electrode voltage-clamp experiments revealed that hCx46wt formed voltage-sensitive hemichannels. This was not observed in the oocytes expressing hCx46D3Y. The replacement of the aspartic acid residue at the third position by another negatively charged residue, glutamic acid, to generate the mutant hCx46D3E, restored the voltage sensitivity of the resultant hemichannels. Moreover, HeLa cell pairs expressing hCx46D3E and hCx46wt showed a similar degree of dye coupling. These results indicate that the negatively charged aspartic acid residue at the third position of the N-terminus of hCx46 could be involved in the determination of the degree of metabolite cell-to-cell coupling and is essential for the voltage sensitivity of the hCx46 hemichannels.     

Distribution of Connexin50 Channels and Hemichannels in Lens Fibers: A Structural Approach

A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques.     

Distribution of connexin50 channels and hemichannels in lens fibers: a structural approach

A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques.     

Structure,Regulation and Function of Gap Junctions in Liver

Abstract

Gap junctions are a specialized group of cell-to-cell junctions that mediate direct intercellular communication between cells. They arise from the interaction of two hemichannels of adjacent cells, which in turn are composed of six connexin proteins. In liver, gap junctions are predominantly found in hepatocytes and play critical roles in virtually all phases of the hepatic life cycle, including cell growth, differentiation, liver-specific functionality and cell death. Liver gap junctions are directed through a broad variety of mechanisms ranging from epigenetic control of connexin expression to post-translational regulation of gap junction activity. This paper reviews established and novel aspects regarding the architecture, control and functional relevance of liver gap junctions.     

Hemichannel and junctional properties of connexin 50

Lens fiber connexins, cx50 and cx46 (alpha3 and alpha8), belong to a small subset of connexins that can form functional hemichannels in nonjunctional membranes. Knockout of either cx50 or cx46 results in a cataract, so the properties of both connexins are likely essential for proper physiological functioning of the lens. Although portions of the sequences of these two connexins are nearly identical, their hemichannel properties are quite different. Cx50 hemichannels are much more sensitive to extracellular acidification than cx46 hemichannels and differ from cx46 hemichannels both in steady-state and kinetic properties. Comparison of the two branches of the cx50 hemichannel G-V curve with the junctional G-V curve suggests that cx50 gap junctions gate with positive relative polarity. The histidine-modifying reagent, diethyl pyrocarbonate, reversibly blocks cx50 hemichannel currents but not cx46 hemichannel currents. Because cx46 and cx50 have very similar amino acid sequences, one might expect that replacing the two histidines unique to the third transmembrane region of cx50 with the corresponding cx46 residues would produce mutants more closely resembling cx46. In fact this does not happen. Instead the mutant cx50H161N does not form detectable hemichannels but forms gap junctions indistinguishable from wild type. Cx50H176Q is oocyte lethal, and the double mutant, cx50H61N/H176Q, neither forms hemichannels nor kills oocytes.     

Disruption in Connexin-Based Communication Is Associated with Intracellular Ca2+ Signal Alterations in Astrocytes from Niemann-Pick Type C Mice

Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1^−/−) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1^−/− hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1^−/− astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1^−/− astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.     

Loss of Gap Junction Plaques and Inhibition of Intercellular Communication in Ilimaquinone-treated BICR-M1Rk and NRK Cells

To examine the mechanism(s) and pathways of gap junction formation and removal a novel and reversible inhibitor of protein secretion, ilimaquinone (IQ), was employed. IQ has been reported to cause the vesiculation of Golgi membranes, block protein transport at the cis-Golgi and depolymerize cytoplasmic microtubules. Connexin43 (Cx43) immunolabeling and dye microinjection experiments revealed that gap junction plaques were lost and intercellular communication was inhibited following IQ treatment for 1 hr in BICR-M1R_k rat mammary tumor cells and for 2 hr in normal rat kidney (NRK) cells. Gap junction plaques and intercellular communication recovered within 2 hr when IQ was removed. IQ, however, did not affect the distribution of zonula occludens-1, a protein associated with tight junctions. Western blot analysis revealed that the IQ-induced loss of gap junction plaques was accompanied by a limited reduction in the highly phosphorylated form of Cx43, previously shown to be correlated with gap junction plaques. The presence of IQ inhibited the formation of new gap junction plaques in BICR-M1R_k cells under conditions where preexisting gap junctions were downregulated by brefeldin A treatment. Treatment of BICR-M1R_k and NRK cells with other microtubule depolymerization agents did not inhibit plaque formation or promote rapid gap junction removal. These findings suggest that IQ disrupts intercellular communication by inhibiting the events that are involved in plaque formation and/or retention at the cell surface independent of its effects on microtubules. Our results also suggest that additional factors other than phosphorylation are necessary for Cx43 assembly into gap junction plaques. Received: 16 January 1996/Revised: 20 September 1996     

Connexin46 Is Retained as Monomers in a trans-Golgi Compartment of Osteoblastic Cells

Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100–solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.     

Changes in Cellular Distribution of Connexins 32 and 26 during Formation of Gap Junctions in Primary Cultures of Rat Hepatocytes

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sectionsin vivo.Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.