Regulation of Connexin43 Oligomerization is Saturable

We have used connexin constructs containing a C-terminal di-lysine-based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, we found that Cx43-HKKSL stably expressed at moderate levels by HeLa cells was retained in the ER and prevented from oligomerization. However, Cx43-HKKSL stably overexpressed by HeLa cells escaped from the ER and localized to a perinuclear region of the cell that included the Golgi apparatus. Overexpressed Cx43-HKKSL oligomerized into hexamers and also formed Triton X-100 insoluble, intracellular complexes that resembled gap junctions. Thus, the ability of HeLa cells to inhibit Cx43 oligomerization was saturable. HeLa cells stably overexpressing Cx43-HKKSL may provide a useful model system to evaluate pharmacologic agents and/or cDNAs encoding chaperones with the potential to regulate initial steps in Cx43 oligomerization.     

Differential oligomerization of endoplasmic reticulum-retained connexin43/connexin32 chimeras

To examine early events in connexin oligomerization, we made connexin constructs containing a C-terminal di-lysine based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL). Previously, we found that both Cx32-HKKSL and Cx43-HKKSL were retained in the ER. However, Cx32-HKKSL oligomerized into hexameric hemichannels, but Cx43-HKKSL was retained as an apparent monomer. To define elements that prevent Cx43-HKKSL oligomerization in the ER, we made a series of HKKSL-tagged Cx43/Cx32 chimeras. When expressed by HeLa cells, some chimeras were retained in the ER as apparent monomers, whereas others oligomerized in the ER. To date, the second and third transmembrane domains and the cytoplasmic loop domain provide the minimal sufficient Cx43 element to inhibit ER oligomerization.     

Differential Oligomerization of Endoplasmic Reticulum-Retained Connexin43/Connexin32 Chimeras

To examine early events in connexin oligomerization, we made connexin constructs containing a C-terminal di-lysine based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL). Previously, we found that both Cx32-HKKSL and Cx43-HKKSL were retained in the ER. However, Cx32-HKKSL oligomerized into hexameric hemichannels, but Cx43-HKKSL was retained as an apparent monomer. To define elements that prevent Cx43-HKKSL oligomerization in the ER, we made a series of HKKSL-tagged Cx43/Cx32 chimeras. When expressed by HeLa cells, some chimeras were retained in the ER as apparent monomers, whereas others oligomerized in the ER. To date, the second and third transmembrane domains and the cytoplasmic loop domain provide the minimal sufficient Cx43 element to inhibit ER oligomerization.     

Targeted gap junction protein constructs reveal connexin-specific differences in oligomerization

To define further the mechanisms of gap junction protein (connexin (Cx)) oligomerization without pharmacologic disruption, we have examined the transport and assembly of connexin constructs containing C-terminal di-lysine-based endoplasmic reticulum (ER) (HKKSL) or ER-Golgi intermediate compartment (AKKFF) targeting sequences. By immunofluorescence microscopy, Cx43-HKKSL transiently transfected into HeLa cells showed a predominantly ER localization, although Cx43-AKKFF was localized to the perinuclear region of the cell. Sucrose gradient analysis of Triton X-100-solubilized connexins showed that either Cx43-HKKSL or Cx43-AKKFF expressed alone by HeLa cells was maintained as an apparent monomer. In contrast to Cx43-HKKSL, Cx32-HKKSL was maintained in the ER as stable hexamers, consistent with the notion that Cx32 and Cx43 oligomerization occur in distinct intracellular compartments. Furthermore, Cx43-HKKSL and Cx43-AKKFF inhibited trafficking of Cx43 and Cx46 to the plasma membrane. The inhibitory effect was because of the formation of mixed oligomers between Cx43-HKKSL or Cx43-AKKF and wild type Cx43 or Cx46. Taken together, these results suggest that Cx43-HKKSL and Cx43-AKKFF recirculate through compartments where oligomerization occurs and may be maintained as apparent monomers by a putative Cx43-specific quality control mechanism.     

Identification of rab20 as a potential regulator of connexin 43 trafficking

Connexin oligomerization and trafficking are regulated processes. To identify proteins that control connexin 43 (Cx43), a screen was designed using HeLa cells expressing a Cx43 construct with di-lysine endoplasmic reticulum (ER)-retention/retrieval motif, Cx43-HKKSL. At moderate levels of expression, Cx43-HKKSL is retained in the ER as monomers; however, Cx43-HKKSL stably overexpressed by HeLa cells localizes to the perinuclear region and oligomerizes. HeLa/Cx43-HKKSL overexpressors were transiently transfected with pooled clones from a human kidney cDNA library and used immunofluorescence microscopy to identify cDNAs that enabled overexpressed Cx43-HKKSL to convert from a perinuclear to ER localization pattern. Using this approach, a small molecular weight GTPase, rab20, was identified as a candidate protein with the ability to regulate Cx43 trafficking. Enhanced green fluorescent protein (EGFP)-tagged rab20 showed a predominantly perinuclear and ER localization pattern and caused wild-type Cx43 to be retained inside the cell. By contrast, mutant EGFP-rab20T19N, which lacks the ability to bind GTP, had no effect on Cx43. These results suggest Cx43 is transported through an intracellular compartment regulated by rab20 along the secretory pathway.     

Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum

In contrast to most multimeric transmembrane complexes that oligomerize in the endoplasmic reticulum (ER), the gap junction protein connexin43 (Cx43) oligomerizes in an aspect of the Golgi apparatus. The mechanisms that prevent oligomerization of Cx43 and related connexins in the ER are not well understood. Also, some studies suggest that connexins can oligomerize in the ER. We used connexin constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, Cx43-HKKSL was retained in the ER and prevented from oligomerization. However, another ER-retained HKKSL-tagged connexin, Cx32-HKKSL, had the capacity to oligomerize. Because this suggested that Cx43 contains a motif that prevented oligomerization in the ER, a series of HKKSL-tagged and untagged Cx32/Cx43 chimeras was screened to define this motif. The minimal motif, which prevented ER oligomerization, consisted of the complete third transmembrane domain and the second extracellular loop from Cx43 on a Cx32 backbone. We propose that charged residues present in Cx43 and related connexins help prevent ER oligomerization by stabilizing the third transmembrane domain in the membrane bilayer.     

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.     

Cytoplasmic Amino Acids within the Membrane Interface Region Influence Connexin Oligomerization

Gap junction channels composed of connexins connect cells, allowing intercellular communication. Their cellular assembly involves a unique quality-control pathway. Some connexins [including connexin43 (Cx43) and Cx46] oligomerize in the trans-Golgi network following export of stabilized monomers from the endoplasmic reticulum (ER). In contrast, other connexins (e.g., Cx32) oligomerize early in the secretory pathway. Amino acids near the cytoplasmic aspect of the third transmembrane domain have previously been shown to determine this difference in assembly sites. Here, we characterized the oligomerization of two connexins expressed prominently in the vasculature, Cx37 and Cx40, using constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) or treatment with brefeldin A to block ER vesicle trafficking. Both methods led to intracellular retention of connexins, since the cells lacked gap junction plaques. Retention of Cx40 in the ER prevented it from oligomerizing, comparable to Cx43. By contrast, ER-retained Cx37 was partially oligomerized. Replacement of two amino acids near the third transmembrane domain of Cx43 (L152 and R153) with the corresponding amino acids from Cx37 (M152 and G153) resulted in early oligomerization in the ER. Thus, residues that allow Cx37 to oligomerize early in the secretory pathway could restrict its interactions with coexpressed Cx40 or Cx43 by favoring homomeric oligomerization, providing a structural basis for cells to produce gap junction channels with different connexin composition.     

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.     

Gap junction channels formed by coexpressed connexin40 and connexin43

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.     

Heterocellular gap junctional communication between alveolar epithelial cells

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.     

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.     

Incompatibility of connexin 40 and 43 Hemichannels in gap junctions between mammalian cells is determined by intracellular domains.

Murine connexin 40 (Cx40) and connexin 43 (Cx43) do not form functional heterotypic gap junction channels. This property may contribute to the preferential propagation of action potentials in murine conductive myocardium (expressing Cx40) which is surrounded by working myocardium, expressing Cx43. When mouse Cx40 and Cx43 were individually expressed in cocultured human HeLa cells, no punctate immunofluorescent signals were detected on apposed plasma membranes between different transfectants, using antibodies specific for each connexin, suggesting that Cx40 and Cx43 hemichannels do not dock to each other. We wanted to identify domains in these connexin proteins which are responsible for the incompatibility. Thus, we expressed in HeLa cells several chimeric gene constructs in which different extracellular and intracellular domains of Cx43 had been spliced into the corresponding regions of Cx40. We found that exchange of both extracellular loops (E1 and E2) in this system (Cx40*43E1,2) was required for formation of homotypic and heterotypic conductive channels, although the electrical properties differed from those of Cx40 or Cx43 channels. Thus, the extracellular domains of Cx43 can be directed to form functional homo- and heterotypic channels. Another chimeric construct in which both extracellular domains and the central cytoplasmic loop (E1, E2, and C2) of Cx43 were spliced into Cx40 (Cx40*43E1,2,C2) led to heterotypic coupling only with Cx43 and not with Cx40 transfectants. Thus, the central cytoplasmic loop of Cx43 contributed to selectivity. A third construct, in which only the C-terminal domain (C3) of Cx43 was spliced into Cx40, i.e., Cx40*43C3, showed neither homotypic nor heterotypic coupling with Cx40 and Cx43 transfectants, suggesting that the C-terminal region of Cx43 determined incompatibility.     

Intracellular calcium regulation of connexin43

The mechanism by which intracellular Ca(2+) concentration ([Ca(2+)](i)) regulates the permeability of gap junctions composed of connexin43 (Cx43) was investigated in HeLa cells stably transfected with this connexin. Extracellular addition of Ca(2+) in the presence of the Ca(2+) ionophore ionomycin produced a sustained elevation in [Ca(2+)](i) that resulted in an inhibition of the cell-to-cell transfer of the fluorescent dye Alexa fluor 594 (IC(50) of 360 nM Ca(2+)). The Ca(2+) dependency of this inhibition of Cx43 gap junctional permeability is very similar to that described in sheep lens epithelial cell cultures that express the three sheep lens connexins (Cx43, Cx44, and Cx49). The intracellular Ca(2+)-mediated decrease in cell-to-cell dye transfer was prevented by an inhibitor of calmodulin action but not by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II or protein kinase C. In experiments that used HeLa cells transfected with a Cx43 COOH-terminus truncation mutant (Cx43(Delta257)), cell-to-cell coupling was similarly decreased by an elevation of [Ca(2+)](i) (IC(50) of 310 nM Ca(2+)) and similarly prevented by the addition of an inhibitor of calmodulin. These data indicate that physiological concentrations of [Ca(2+)](i) regulate the permeability of Cx43 in a calmodulin-dependent manner that does not require the major portion of the COOH terminus of Cx43.     

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     

Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells.

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin-Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 microm and 0.5-1.5 microm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.     

Enhancement of connexin 43 expression increases proliferation and differentiation of an osteoblast-like cell line

Bone cells form a functional syncytium as they are coupled by gap junctions composed mainly of connexin 43 (Cx43). To further understand the role of Cx43 in bone cell growth and differentiation, we stably transfected Cx45-expressing UMR 106-01 cells with Cx43 using an expression vector containing rat Cx43 cDNA. Three stably transfected clones were analyzed, all of which showed altered expression of Cx43 and/or Cx45 as was obvious from immunocytochemistry and Northern blotting. Double whole-cell patch clamping revealed single-channel conductances of 20 (Cx45) and 60 pS (Cx43). The overexpression of Cx43 led to an increase in dye coupling concomitant with elevated gap-junctional conductance. The phenotype of the transfected clones was characterized by an increased proliferation (4- to 7-fold) compared to controls. Moreover, a transfectant clone with 10- to 12-fold enhanced Cx43 expression showed a significantly increased calcium content of the extracellular matrix and enlarged mineralization nodules, while alkaline phosphatase was moderately increased. We conclude that enhanced gap-junctional coupling via Cx43 significantly promotes proliferation and differentiation of UMR cells.     

Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.     

Tetracysteine Genetic Tags Complexed with Biarsenical Ligands as a Tool for Investigating Gap Junction Structure and Dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.