Mutational analysis of gap junction formation.

The paired oocyte cell-cell channel assay was used to investigate the mechanisms involved in the process of formation of gap junction channels. Single oocytes, injected with connexin-specific mRNAs, accumulate a pool of precursors from which cell-cell channels can form rapidly upon pairing. Several lines of evidence, including immunohistochemistry and surface labeling, indicate that part of this precursor pool is located in the cell membrane, probably in the form of closed hemichannels. The homophilic binding of hemichannels to each other can be mimicked by synthetic peptides representing the extracellular loop sequences of connexin32. The peptides specifically suppress channel formation. A crucial role is established for the six cysteines in the extracellular domains that are conserved in all vertebrate gap junction proteins. Change of any of these cysteines into serines results in absolute loss of function of the mutant connexin. The effects of thiol-specific reagents on channel formation suggest that docking and/or opening of channels involves disulfide exchange. Several of the variable amino acids in the extracellular loop sequences were found to determine specificity of connexin-connexin interactions.     

Gap junction-mimetic peptides do work, but in unexpected ways

Gap junction mimetic peptides containing sequences of the extracellular loops of connexins inhibit the de-novo formation of gap junction channels but do not impair the function of existing cell-cell channels. Recently, a flurry of publications appeared showing that such “GAP” peptides attenuate ATP release and/or surrogate measures of it. Although no direct effect on putative connexin “hemichannels” has ever been shown, the peptide effect has been used as diagnostic tool for demonstrating the existence of such channels. However, testing of the peptides on genuine unapposed membrane channels formed by connexins failed to reveal any inhibitory action of the peptides on channel activity. Instead, membrane channels formed by the unrelated pannexin1 were inhibited in the same concentration range as described for the release of ATP. Consequently, rather than indicating connexin involvement in ATP release, the GAP peptide effects represent supporting evidence for a role of pannexin1 in this process.     

Mimetic peptides as blockers of connexin channel-facilitated intercellular communication

There is a dearth of chemical inhibitors of connexin-mediated intercellular communication. The advent of short “designer” connexin mimetic peptides has provided new tools to inhibit connexin channels quickly and reversibly. This perspective describes the development of mimetic peptides, especially Gap 26 and 27 that are the most popular and correspond to specific sequences in the extracellular loops of connexins 37, 40 and 43. Initially they were used to inhibit gap-junctional coupling in a wide range of mammalian cells and tissues. Currently, they are also being examined as therapeutic agents that accelerate wound healing and in the early treatment of spinal cord injury. The mimetic peptides bind to connexin hemichannels, influencing channel properties as shown by lowering of electrical conductivity and potently blocking the entry of small reporter dyes and the release of ATP by cells. A mechanism is proposed to help explain the dual action of connexin mimetic peptides on connexin hemichannels and gap-junctional coupling.     

The Pattern of Disulfide Linkages in the Extracellular Loop Regions of Connexin 32 Suggests a Model for the Docking Interface of Gap Junctions

Connexins, like true cell adhesion molecules, have extracellular domains that provide strong and specific homophilic, and in some cases, heterophilic interactions between cells. Though the structure of the binding domains of adhesion proteins have been determined, the extracellular domains of connexins, consisting of two loops of ~34–37 amino acids each, are not easily studied in isolation from the rest of the molecule. As an alternative, we used a novel application of site-directed mutagenesis in which four of the six conserved cysteines in the extracellular loops of connexin 32 were moved individually and in all possible pairwise and some quadruple combinations. This mapping allowed us to deduce that all disulfides form between the two loops of a single connexin, with the first cysteine in one loop connected to the third of the other. Furthermore, the periodicity of movements that produced functional channels indicated that these loops are likely to form antiparallel β sheets. A possible model that could explain how these domains from apposed connexins interact to form a complete channel is discussed.     

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.     

Two-dimensional kinetics of inter-connexin interactions from single-molecule force spectroscopy

Gap junction channels are intercellular channels that form by docking the extracellular loops of connexin protein subunits. While the structure and function of gap junctions as intercellular channels have been characterized using different techniques, the physics of the inter-connexin interaction remain unknown. Moreover, as far as we know, the capacity of gap junction channels to work as adhesion complexes supporting pulling forces has not yet been quantitatively addressed. We report the first quantitative characterization of the kinetics and binding strength of the interaction of a short peptide mimicking extracellular loop 2 of Cx26 with membrane-reconstituted Cx26, combining the imaging and force spectroscopy capabilities of atomic force microscopy. The fast dissociation rate inferred a dynamic bond, while the slow association rate reflected the reduced flexibility and small size of extracellular loops. Our results propose the gap junction channel as an adhesion complex that associates slowly and dissociates fast at low force but is able to support important pulling forces in its native, hexameric form.     

Cell/cell channel formation involves disulfide exchange.

The oocyte cell/cell-channel assay was used to identify amino acids involved in the process of cell/cell-channel formation. The expression of the rat liver gap-junction protein, connexin 32, in single oocytes, results in the accumulation of a pool of channel precursors. Upon pairing of such oocytes, cell/cell channels form rapidly from this pool. The rate of formation is affected by thiol-specific reagents and the pH. This suggests the involvement of extracellular cysteine residues in the channel formation process. Two connexin-32 mutants were generated by site-directed mutagenesis in which cysteine residues were replaced by serine. Both mutant connexins were unable to form cell/cell channels. Thus, the cysteine residues appear to play an important role in the channel formation process.     

Modulation of membrane channel currents by gap junction protein mimetic peptides: size matters

Connexin mimetic peptides are widely used to assess the contribution of nonjunctional connexin channels in several processes, including ATP release. These peptides are derived from various connexin sequences and have been shown to attenuate processes downstream of the putative channel activity. Yet so far, no documentation of effects of peptides on connexin channels has been presented. We tested several connexin and pannexin mimetic peptides and observed attenuation of channel currents that is not compatible with sequence specific actions of the peptides. Connexin mimetic peptides inhibited pannexin channel currents but not the currents of the channel formed by connexins from which the sequence was derived. Pannexin mimetic peptides did inhibit pannexin channel currents but also the channels formed by connexin 46. The same pattern of effects was observed for dye transfer, except that the inhibition levels were more pronounced than for the currents. The channel inhibition by peptides shares commonalities with channel effects of polyethylene glycol (PEG), suggesting a steric block as a mechanism. PEG accessibility is in the size range expected for the pore of innexin gap junction channels, consistent with a functional relatedness of innexin and pannexin channels. mimetic peptide; polyethylene glycol; calcium wave     

Gating properties of connexin32 cell-cell channels and their mutants expressed in Xenopus oocytes.

Carboxyl-terminal deletion mutants of the gap junction protein connexin32 were tested in the oocyte cell-cell channel assay. Oocytes expressing a mutant lacking 58 carboxyl terminal amino acids were found to exhibit junctional conductances of the same magnitude as oocytes expressing wild-type connexin32. The gating properties of the channels formed by this mutant of connexin32 with respect to transjunctional voltage and cytoplasmic acidification are indistinguishable from those found with wild-type connexin32 channels. This includes a novel pH-dependent voltage gate. In another mutant, two carboxyl terminal serine residues, Ser233 and Ser240, were replaced by Asn residues. This double mutant has properties indistinguishable from wild-type connexin32, suggesting that phosphorylation of either of these serines is not required for channel opening.     

Perturbing plasma membrane hemichannels attenuates calcium signalling in cardiac cells and HeLa cells expressing connexins

Many cell signalling pathways are driven by changes in cytosolic calcium. We studied the effects of a range of inhibitors of connexin channels on calcium signalling in cardiac cells and HeLa cells expressing connexins. Gap 26 and 27, peptides that mimic short sequences in each of the extracellular loops of connexin 43, and anti-peptide antibodies generated to extracellular loop sequences of connexins, inhibited calcium oscillations in neonatal cardiac myocytes, as well as calcium transients induced by ATP in HL-1 cells originating from cardiac atrium and HeLa cells expressing connexin 43 or 26. Comparison of single with confluent cells showed that intracellular calcium responses were suppressed by interaction of connexin mimetic peptides and antibodies with hemichannels present on unapposed regions of the plasma membrane. To investigate how inhibition of hemichannels in the plasma membrane by the applied reagents was communicated to calcium store operation in the endoplasmic reticulum, we studied the effect of Gap 26 on calcium entry into cells and on intracellular IP3 release; both were inhibited by Gap 26. Calcium transients in both connexin 43- and connexin 26-expressing HeLa cells were inhibited by the peptides suggesting that the extended cytoplasmic carboxyl tail domain of larger connexins and their interactions with intracellular scaffolding/auxiliary proteins were unlikely to feature in transmitting peptide-induced perturbations at hemichannels in the plasma membrane to IP3 receptor channel central to calcium signalling. The results suggest that calcium levels in a microenvironment functionally connecting plasma membrane connexin hemichannels to downstream IP3-dependent calcium release channels in the endoplasmic reticulum were disrupted by the connexin mimetic peptide, although implication of other candidate hemichannels cannot be entirely discounted. Since calcium signalling is fundamental to the maintenance of cellular homeostasis, connexin hemichannels emerge as therapeutic targets open to manipulation by reagents interacting with external regions of these channels.     

Dynamics of gap junctions observed in living cells with connexin43-GFP chimeric protein

To study the aggregation of cell-to-cell channels into gap junctions at individual cell-cell contacts, we transfected cells with an expression vector for a chimeric protein composed of the cell-to-cell channel protein connexin43 and a green fluorescent protein. The chimeric channel protein was visualized in the fluorescence microscope and was found to form gap junctions at the cell-cell contacts just like wild-type connexin43. Cells expressing the chimeric protein had functional cell-to-cell channels. Using timelapse videomicroscopy on live cells we observed individual gap junctions over long periods and recorded the time course of aggregation of the chimeric channel protein into gap junctions at newly formed cell-cell contacts. We found that individual small gap junctions were very dynamic, moving about or becoming assembled and disassembled in the course of minutes. Larger gap junctions were more stable than small punctate ones. In control condition, stable new gap junctions were not formed during observation times of 30 min or longer. But at elevated levels of cyclic adenosine monophosphate, the chimeric channel protein began aggregating at new junctions 5-10 minutes after cell-cell contact and continued to concentrate there for at least one hour. Also already established junctions grew in size. The fluorescent chimeric channel protein will be an excellent tool to investigate the regulation of trafficking of connexin from and to the membrane and the mechanism of connexin channel aggregation into gap junctions.     

Proteomic analysis of blastema formation in regenerating axolotl limbs

Background

For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood.

Results

Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred.

Conclusion

This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.     

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.     

Mechanism for modulation of gating of connexin26-containing channels by taurine

The mechanisms of action of endogenous modulatory ligands of connexin channels are largely unknown. Previous work showed that protonated aminosulfonates (AS), notably taurine, directly and reversibly inhibit homomeric and heteromeric channels that contain Cx26, a widely distributed connexin, but not homomeric Cx32 channels. The present study investigated the molecular mechanisms of connexin channel modulation by taurine, using hemichannels and junctional channels composed of Cx26 (homomeric) and Cx26/Cx32 (heteromeric). The addition of a 28-amino acid "tag" to the carboxyl-terminal domain (CT) of Cx26 (Cx26(T)) eliminated taurine sensitivity of homomeric and heteromeric hemichannels in cells and liposomes. Cleavage of all but four residues of the tag (Cx26(Tc)) resulted in taurine-induced pore narrowing in homomeric hemichannels, and restored taurine inhibition of heteromeric hemichannels (Cx26(Tc)/Cx32). Taurine actions on junctional channels were fully consistent with those on hemichannels. Taurine-induced inhibition of Cx26/Cx32(T) and nontagged Cx26 junctional channels was blocked by extracellular HEPES, a blocker of the taurine transporter, confirming that the taurine-sensitive site of Cx26 is cytoplasmic. Nuclear magnetic resonance of peptides corresponding to Cx26 cytoplasmic domains showed that taurine binds to the cytoplasmic loop (CL) and not the CT, and that the CT and CL directly interact. ELISA showed that taurine disrupts a pH-dependent interaction between the CT and the CT-proximal half of the CL. These studies reveal that AS disrupt a pH-driven cytoplasmic interdomain interaction in Cx26-containing channels, causing closure, and that the Cx26CT has a modulatory role in Cx26 function.     

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.     

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.     

Inhibition of thyrotropin binding to receptor by synthetic human thyrotropin beta peptides

In order to study the structure and function relationships of the thyrotropin (TSH)-specific beta-subunit, we produced 11 synthetic overlapping peptides containing the entire 112-amino acid sequence of human beta TSH and tested them for activity in TSH radioreceptor assay using both human and porcine thyroid membranes. Synthetic peptides representing four regions of the beta-subunit demonstrated the ability to inhibit binding of 125I-bovine TSH to crude thyroid membranes. The peptide representing the -COOH terminus of the subunit (beta 101-112) possessed highest binding activity, inhibiting binding of labeled TSH with an EC50 of 80 microM. The remaining active peptides were: beta 71-85 (104 microM), beta 31-45 (186 microM), beta 41-55 (242 microM), and beta 1-15 (331 microM). Specificity of the binding activity was shown by the inability of the peptides representing the remainder of the subunit to inhibit binding of label and by the inability of any of the peptides to inhibit binding of 125I-epidermal growth factor to the same thyroid membranes. The low affinity of the peptides as compared with native hormone is in agreement with previous studies of synthetic alpha-subunit peptides and, further, suggests that the interaction of beta TSH with receptor is multifaceted, requiring cooperative binding of these sites for the observed high affinity of the whole hormone. These studies are in agreement with previous predictions of active regions by chemical modification but add two regions to the list, showing the utility of the synthetic peptide strategy in the study of peptide hormone structure-activity relationships.     

Aminosulfonate modulated pH-induced conformational changes in connexin26 hemichannels

Gap junction channels regulate cell-cell communication by passing metabolites, ions, and signaling molecules. Gap junction channel closure in cells by acidification is well documented; however, it is unknown whether acidification affects connexins or modulating proteins or compounds that in turn act on connexins. Protonated aminosulfonates directly inhibit connexin channel activity in an isoform-specific manner as shown in previously published studies. High-resolution atomic force microscopy of force-dissected connexin26 gap junctions revealed that in HEPES buffer, the pore was closed at pH < 6.5 and opened reversibly by increasing the pH to 7.6. This pH effect was not observed in non-aminosulfonate buffers. Increasing the protonated HEPES concentration did not close the pore, indicating that a saturation of the binding sites occurs at 10 mM HEPES. Analysis of the extracellular surface topographs reveals that the pore diameter increases gradually with pH. The outer connexon diameter remains unchanged, and there is a approximately 6.5 degrees rotation in connexon lobes. These observations suggest that the underlying mechanism closing the pore is different from an observed Ca2+-induced closure.