Intramolecular interactions mediate pH regulation of connexin43 channels.

We have previously proposed that acidification-induced regulation of the cardiac gap junction protein connexin43 (Cx43) may be modeled as a particle-receptor interaction between two separate domains of Cx43: the carboxyl terminal (acting as a particle), and a region including histidine 95 (acting as a receptor). Accordingly, intracellular acidification would lead to particle-receptor binding, thus closing the channel. A premise of the model is that the particle can bind its receptor, even if the particle is not covalently bound to the rest of the protein. The latter hypothesis was tested in antisense-injected Xenopus oocyte pairs coexpressing mRNA for a pH-insensitive Cx43 mutant truncated at amino acid 257 (i.e., M257) and mRNA coding for the carboxyl terminal region (residues 259-382). Intracellular pH (pHo) was recorded using the dextran form of the proton-sensitive dye seminaphthorhodafluor (SNARF). Junctional conductance (Gj) was measured with the dual voltage clamp technique. Wild-type Cx43 channels showed their characteristic pH sensitivity. M257 channels were not pH sensitive (pHo tested: 7.2 to 6.4). However, pH sensitivity was restored when the pH-insensitive channel (M257) was coexpressed with mRNA coding for the carboxyl terminal. Furthermore, coexpression of the carboxyl terminal of Cx43 enhanced the pH sensitivity of an otherwise less pH-sensitive connexin (Cx32). These data are consistent with a model of intramolecular interactions in which the carboxyl terminal acts as an independent domain that, under the appropriate conditions, binds to a separate region of the protein and closes the channel. These interactions may be direct (as in the ball-and-chain mechanism of voltage-dependent gating of potassium channels) or mediated through an intermediary molecule. The data further suggest that the region of Cx43 that acts as a receptor for the particle is conserved among connexins. A similar molecular mechanism may mediate chemical regulation of other channel proteins.     

Association with ZO-1 Correlates with Plasma Membrane Partitioning in Truncated Connexin45 Mutants

Zonula occludens-1 (ZO-1), the most abundant known connexin-interacting protein in osteoblastic cells, associates with the carboxyl termini of both Cx43 and Cx45. To learn more about the role of the cormexin-ZO-1 interaction, we analyzed connexin trafficking and function in ROS 17/2.8 cells that were stably transfected either with full length Cx45 or with Cx45 lacking 34 or 37 amino acids on the carboxyl terminus (Cx45t34 or Cx45t37). All three proteins were transported to appositional membranes in the transfected cells: Cx45 and Cx45t34 displayed a punctate appositional membrane-staining pattern, while Cx45t37 staining at appositional membranes was more linear. Expression of Cx45 decreased gap junction communication as assayed by dye transfer, while expression of Cx45t34 or Cx45t37 increased the amount of dye transfer seen in these cells. We found that Cx43, Cx45 and Cx45t34 co-precipitated with ZO-1 in these cells, while Cx45t37 did not. We also found that Cx45t37 was much more soluble in 1% Triton X-100 than the other connexins examined. In addition, Cx45t37 migrated to a fraction of lighter buoyant density on sucrose flotation gradients than Cx43, Cx45, ZO-1 and Cx45t34. As ZO-1 is an actin-binding protein, this suggested that the differences in Cx45t37 solubility might be due to a difference between the interaction of gap junctions and the actin cytoskeleton in the ROS/Cx45t37 and in the other transfected ROS cells. To examine this possibility, the transfected ROS cells were stained with fluorescently labeled phalloidin and demonstrated that there was a notable loss of actin stress fibers in the ROS/Cx45t37 cells. These findings suggest that association with ZO-1 alters the plasma membrane localization of Cx45 by removing it from a lipid raft compartment and rendering it Triton-insoluble, presumably by promoting an interaction with the actin cytoskeleton; they also suggest that Cx45 has a complex binding interaction with ZO-1 that involves either an extended carboxyl terminal domain or two distinct binding sites.     

A novel connexin43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43

The degradation of connexin43 (Cx43) has been reported to involve both lysosomal and proteasomal degradation pathways; however, very little is known about the mechanisms regulating these Cx43 degradation pathways. Using yeast two-hybrid, glutathione S-transferase pull-down, and co-immunoprecipitation approaches, we have identified a novel Cx43-interacting protein of approximately 75 kDa, CIP75. Laser confocal microscopy showed that CIP75 is located primarily at the endoplasmic reticulum, as indicated by the calnexin marker, with Cx43 co-localization in this perinuclear region. CIP75 belongs to the UbL (ubiquitin-like)-UBA (ubiquitin-associated) domain-containing protein family with a N-terminal UbL domain and a C-terminal UBA domain. The UBA domain of CIP75 is the main element mediating the interaction with Cx43, whereas the CIP75-interacting region in Cx43 resides in the PY motif and multiphosphorylation sites located between Lys 264 and Asn 302. Interestingly, the UbL domain interacts with the S2/RPN1 and S5a/RPN10 protein subunits of the regulatory 19 S proteasome cap subunit of the 26 S proteasome complex. Overexpression experiments suggested that CIP75 is involved in the turnover of Cx43 as measured by a significant stimulation of Cx43 degradation and reduction in its half-life with the opposite effects on Cx43 degradation observed in small interference RNA knockdown experiments.     

PH regulation of connexin43: molecular analysis of the gating particle.

Gap junction channels allow for the passage of ions and small molecules between neighboring cells. These channels are formed by multimers of an integral membrane protein named connexin. In the heart and other tissues, the most abundant connexin is a 43-kDa, 382-amino acid protein termed connexin43 (Cx43). A characteristic property of connexin channels is that they close upon acidification of the intracellular space. Previous studies have shown that truncation of the carboxyl terminal of Cx43 impairs pH sensitivity. In the present study, we have used a combination of optical, electrophysiological, and molecular biological techniques and the oocyte expression system to further localize the regions of the carboxyl terminal that are involved in pH regulation of Cx43 channels. Our results show that regions 261-300 and 374-382 are essential components of a pH-dependent "gating particle," which is responsible for acidification-induced uncoupling of Cx43-expressing cells. Regions 261-300 and 374-382 seem to be interdependent. The function of region 261-300 may be related to the presence of a poly-proline repeat between amino acids 274 and 285. Furthermore, site-directed mutagenesis studies show that the function of region 374-382 is not directly related to its net balance of charges, although mutation of only one amino acid (aspartate 379) for asparagine impairs pH sensitivity to the same extent as truncation of the carboxyl terminal domain (from amino acid 257). The mutation in which serine 364 is substituted for proline, which has been associated with some cases of cardiac congenital malformations in humans, also disrupts the pH gating of Cx43, although deletion of amino acids 364-373 has no effect on acidification-induced uncoupling. These results provide new insight into the molecular mechanisms responsible for acidification-induced uncoupling of gap junction channels in the heart and in other Cx43-expressing structures.     

Novel rab GAP-like protein, CIP85, interacts with connexin43 and induces its degradation

Gap junctions play critical roles in tissue function and homeostasis. Connexin43 (Cx43) is a major gap junction protein expressed in the mammalian heart and other tissues and may be regulated by its interaction with other cellular proteins. Using the yeast two-hybrid screen, we identified a novel Cx43-interacting protein of 85-kDa, CIP85, which contains a single TBC, SH3, and RUN domain, in addition to a short coiled coil region. Homologues containing this unique combination of domains were found in human, D. melanogaster, and C. elegans. CIP85 mRNA is expressed ubiquitously in mouse and human tissues. In vitro interaction assays and in vivo co-immunoprecipitation experiments confirmed the interaction of endogenous CIP85 with Cx43. In vitro interaction experiments using CIP85 mutants with in-frame deletions of the TBC, SH3, and RUN domains indicated that the SH3 domain of CIP85 is involved in its interaction with Cx43. Conversely, analysis of Cx43 mutants with proline to alanine substitutions in the two proline-rich regions of Cx43 revealed that the P(253)LSP(256) motif is an important determinant of the ability of Cx43 to interact with CIP85. Laser-scanning confocal microscopy showed that CIP85 colocalized with Cx43 at the cell periphery, particularly in areas reminiscent of gap junction plaques. The functional importance of the interaction between CIP85 and Cx43 was suggested by the observation that CIP85 appears to induce the turnover of Cx43 through the lysosomal pathway.     

The connexin43-interacting protein,CIP85, mediates the internalization of connexin43 from the plasma membrane

Abstract

CIP85 was previously identified as a connexin43 (Cx43)-interacting protein that is ubiquitously expressed in multiple mammalian tissues and cell types. The interaction between the SH3 domain of CIP85 and a proline-rich region of Cx43 has previously been associated with an increased rate of Cx43 turnover through lysosomal mechanisms. This report presents biochemical and immunofluorescence evidence that overexpression of CIP85 reduced the presence of Cx43 in gap junction plaques at the plasma membrane. Furthermore, this effect was dependent upon the interaction of CIP85 with Cx43 at the plasma membrane. These results indicate that CIP85 increases Cx43 turnover by accelerating the internalization of Cx43 from the plasma membrane. CIP85 was also observed to interact with clathrin, which suggested a role for CIP85 in the clathrin-mediated internalization of Cx43.     

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     

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.     

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.     

The Cx43-like connexin protein Cx40.8 is differentially localized during fin ontogeny and fin regeneration

Connexins (Cx) are the subunits of gap junctions, membraneous protein channels that permit the exchange of small molecules between adjacent cells. Cx43 is required for cell proliferation in the zebrafish caudal fin. Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration. Here we demonstrate that Cx40.8 exhibits novel differential subcellular localization in vivo, depending on the growth status of the fin. During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration. We next identified a 30 amino acid domain of Cx40.8 responsible for its dynamic localization. One possible explanation for the differential localization is that Cx40.8 contributes to the regulation of Cx43 in vivo, perhaps modifying channel activity during ontogenetic growth. However, we find that the voltage-gating properties of Cx40.8 are similar to Cx43. Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus. We suggest that the dynamic localization of Cx40.8 differentially influences Cx43-dependent cell proliferation during ontogeny and regeneration.     

Effect of charge substitutions at residue his-142 on voltage gating of connexin43 channels

Previous studies indicate that the carboxyl terminal of connexin43 (Cx43CT) is involved in fast transjunctional voltage gating. Separate studies support the notion of an intramolecular association between Cx43CT and a region of the cytoplasmic loop (amino acids 119–144; referred to as “L2”). Structural analysis of L2 shows two α-helical domains, each with a histidine residue in its sequence (H126 and H142). Here, we determined the effect of H142 replacement by lysine, alanine, and glutamate on the voltage gating of Cx43 channels. Mutation H142E led to a significant reduction in the frequency of occurrence of the residual state and a prolongation of dwell open time. Macroscopically, there was a large reduction in the fast component of voltage gating. These results resembled those observed for a mutant lacking the carboxyl terminal (CT) domain. NMR experiments showed that mutation H142E significantly decreased the Cx43CT-L2 interaction and disrupted the secondary structure of L2. Overall, our data support the hypothesis that fast voltage gating involves an intramolecular particle-receptor interaction between CT and L2. Some of the structural constrains of fast voltage gating may be shared with those involved in the chemical gating of Cx43.     

Ubiquitin-independent proteasomal degradation of endoplasmic reticulum-localized connexin43 mediated by CIP75

Connexin43 (Cx43) is a transmembrane protein that forms gap junction channels. Regulation of Cx43 turnover is one mechanism to control the level of intercellular communication that occurs through gap junction channels. Proteasomal degradation of Cx43 is regulated in part through CIP75, a ubiquitin-like and ubiquitin-associated domain containing protein. CIP75 interacts with endoplasmic reticulum-localized Cx43, as demonstrated through co-immunoprecipitation and immunofluorescence microscopy experiments. CIP75 also binds to free monoubiquitin and lysine 48-linked tetraubiquitin chains in vitro and binds to ubiquitinated proteins in cellular lysates. However, analysis of Cx43 that immunoprecipitated with CIP75 demonstrated that the Cx43 associated with CIP75 was not ubiquitinated, and a mutant form of Cx43 that lacked lysines capable of ubiquitination retained the capacity to interact with CIP75. These results suggest that although CIP75 can interact with ubiquitinated cellular proteins, its interaction with Cx43 and stimulation of Cx43 proteasomal degradation does not require the ubiquitination of Cx43.     

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.     

Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31.

A new member of the connexin gene family has been identified and designated rat connexin-31 (Cx31) based on its predicted molecular mass of 30,960 daltons. Cx31 is 270 amino acids long and is coded for by a single copy gene. It is expressed as a 1.7-kilobase mRNA that is detected in placenta, Harderian gland, skin, and eye. Cx31 is highly conserved and can be detected in species as distantly related to rat as Xenopus laevis. It exhibits extensive sequence similarity to the previously identified connexins, 58, 50, and 40% amino acid identity to Cx26, Cx32, and Cx43, respectively. When conservation of predicted phosphorylation sites is used to adjust the alignment of Cx31 to other connexins, a unique alignment of three predicted protein kinase C phosphorylation sites near the carboxyl terminus of Cx31 with three sites at the carboxyl terminus of Cx43 is revealed.     

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.     

pH-dependent dimerization of the carboxyl terminal domain of Cx43

Previous studies have demonstrated that the carboxyl terminus of the gap junction protein Cx43 (Cx43CT) can act as an independent, regulatory domain that modulates intercellular communication in response to appropriate chemical stimuli. Here, we have used NMR, chemical cross-linking, and analytical ultracentrifugation to further characterize the biochemical and biophysical properties of the Connexin43 carboxyl terminal domain (S255-I382). NMR-diffusion experiments at pH 5.8 suggested that the Connexin43 carboxyl terminus (CX43CT) may have a molecular weight greater than that of a monomer. Sedimentation equilibrium and cross-linking data demonstrated a predominantly dimeric state for the Cx43CT at pH 5.8 and 6.5, with limited dimer formation at a more neutral pH. NMR-filtered nuclear Overhauser effect studies confirmed these observations and identified specific areas of parallel orientation within Cx43CT, likely corresponding to dimerization domains. These regions included a portion of the SH3 binding domain, as well as two fragments previously found to organize in alpha-helical structures. Together, these data show that acidification causes Cx43CT dimer formation in vitro. Whether dimer formation is an important structural component of the regulation of Connexin43 channels remains to be determined. Dimerization may alter the affinity of Cx43CT regions for specific molecular partners, thus modifying the regulation of gap junction channels.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Fusion of GFP to the carboxyl terminus of connexin43 increases gap junction size in HeLa cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.