Structure and closure of connexin gap junction channels

Connexin gap junctions comprise assembled channels penetrating two plasma membranes for which gating regulation is associated with a variety of factors, including voltage, pH, Ca²⁺, and phosphorylation. Functional studies have established that various parts of the connexin peptides are related to channel closure and electrophysiology studies have provided several working models for channel gating. The corresponding structural models supporting these findings, however, are not sufficient because only small numbers of closed connexin structures have been reported. To fully understand the gating mechanisms, the channels should be visualized in both the open and closed states. Electron crystallography and X-ray crystallography studies recently revealed three-dimensional structures of connexin channels in a couple of states in which the main difference is the conformation of the N-terminal domain, which have helped to clarify the structure in regard to channel closure. Here the closure models for connexin gap junction channels inferred from structural and functional studies are described in the context of each domain of the connexin protein associated with gating modulation.     

Mix and match: Investigating heteromeric and heterotypic gap junction channels in model systems and native tissues

This review is based in part on a roundtable discussion session: “Physiological roles for heterotypic/heteromeric channels” at the 2013 International Gap Junction Conference (IGJC 2013) in Charleston, South Carolina. It is well recognized that multiple connexins can specifically co-assemble to form mixed gap junction channels with unique properties as a means to regulate intercellular communication. Compatibility determinants for both heteromeric and heterotypic gap junction channel formation have been identified and associated with specific connexin amino acid motifs. Hetero-oligomerization is also a regulated process; differences in connexin quality control and monomer stability are likely to play integral roles to control interactions between compatible connexins. Gap junctions in oligodendrocyte:astrocyte communication and in the cardiovascular system have emerged as key systems where heterotypic and heteromeric channels have unique physiologic roles. There are several methodologies to study heteromeric and heterotypic channels that are best applied to either heterologous expression systems, native tissues or both. There remains a need to use and develop different experimental approaches in order to understand the prevalence and roles for mixed gap junction channels in human physiology.     

Molecular organization of gap junction membrane channels

Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas formingin vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.     

Characterization of the gap junction protein,connexin45

Connexin45 is a gap junction protein which forms channels with unique characteristics. RNA blots demonstrated that connexin45 is expressed in a number of cell lines including WB, SK Hepl, BHK, A7r5, CLEM, and BWEM cells. Connexin45 was further studied in BWEM cells using specific affinity-purified antibodies directed against a synthetic peptide representing amino acids 285–298 of its sequence. Immunofluorescence experiments demonstrated that the BWEM cells expressed both connexin43 and connexin45 and that these connexins colocalized. Connexin45 polypeptide, immunoprecipitated from BWEM cells metabolically labeled with [³⁵S]-methionine, consisted of a predominant 48 kD polypeptide. Connexin45 and connexin43 contained radioactive phosphate when immunoprecipitated from BWEM cells metabolically labeled with [³²P]-orthophosphoric acid. This phosphate label was removed from connexin45 by alkaline phosphatase digestion. Treatment of BWEM cells with the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited intercellular passage of microinjected Lucifer yellow. While TPA treatment induced phosphorylation of connexin43 in these cells, it reduced the expression of connexin45. Furthermore, the connexin45 expressed after TPA treatment was not phosphorylated. These results suggest that treatments which alter protein phosphorylation may regulate connexin43 and connexin45 in BWEM cells by different mechanisms.These studies were supported by National Institutes of Health grants HL45466 and EY08368. J.G.L. is supported by a fellowship from the Lucille P. Markey Foundation. E.C.B. is an Established Investigator of the American Heart Association.     

Cx23, a connexin with only four extracellular-loop cysteines, forms functional gap junction channels and hemichannels

Gap junction channels may be comprised of either connexin or pannexin proteins (innexins and pannexins). Membrane topologies of both families are similar, but sequence similarity is lacking. Recently, connexin-like sequences have been identified in mammalian and zebrafish genomes that have only four conserved cysteines in the extracellular domains (Cx23), a feature of the pannexins. Phylogenetic analyses of the non-canonical "C4" connexins reveal that these sequences are indeed connexins. Functional assays reveal that the Cx23 gap junctions are capable of sharing neurobiotin, and further, that Cx23 connexins form hemichannels in vitro.     

Role of intramolecular interaction in connexin50: mediating the Ca2+-dependent binding of calmodulin to gap junction

Gap junction channels formed by connexin50 (Cx50) are critical for maintenance of eye lens transparency. Cleavage of the carboxyl terminus (CT) of Cx50 to produce truncated Cx50 (Cx50trunc) occurred naturally during maturation of lens fiber cells. The mechanism of its altered properties is under confirmation. It has been suggested that calmodulin (CaM) participates in gating some kinds of gap junction. Here, we performed confocal colocalization and co-immunoprecipitation experiments to study the relationships between Cx50 and CaM. Results exhibited that the CaM could colocalize Ca2+ dependently with CT in the linear area of cell-to-cell contact formed by Cx50trunc, while it could not localize in the linear area without expression of CT. Further study indicated that the CT could interact Ca2+ independently with the cytoplasmic loop (CL) of Cx50. These data put forward the importance of Ca2+-independent intramolecular interaction between CT and CL of Cx50, which mediate the Ca2+-dependent binding of CaM to Cx50. These intra- and intermolecular interactions may further improve our understanding of biological significance of the Cx50 in the eye lens.     

Double-membrane gap junction internalization requires the clathrin-mediated endocytic machinery

Direct cell-cell communication mediated by plasma membrane-spanning gap junction (GJ) channels is vital to all aspects of cellular life. Obviously, GJ intercellular communication (GJIC) requires precise regulation, and it is known that controlled biosynthesis and degradation, and channel opening and closing (gating) are exploited. We discovered that cells internalize GJs in response to various stimuli. Here, we report that GJ internalization is a clathrin-mediated endocytic process that utilizes the vesicle-coat protein clathrin, the adaptor proteins adaptor protein complex 2 and disabled 2, and the GTPase dynamin. To our knowledge, we are first to report that the endocytic clathrin machinery can internalize double-membrane vesicles into cells.     

Structural organization of gap junction channels

Gap junctions were initially described morphologically, and identified as semi-crystalline arrays of channels linking two cells. This suggested that they may represent an amenable target for electron and X-ray crystallographic studies in much the same way that bacteriorhodopsin has. Over 30 years later, however, an atomic resolution structural solution of these unique intercellular pores is still lacking due to many challenges faced in obtaining high expression levels and purification of these structures. A variety of microscopic techniques, as well as NMR structure determination of fragments of the protein, have now provided clearer and correlated views of how these structures are assembled and function as intercellular conduits. As a complement to these structural approaches, a variety of mutagenic studies linking structure and function have now allowed molecular details to be superimposed on these lower resolution structures, so that a clearer image of pore architecture and its modes of regulation are beginning to emerge.     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

Calcium-dependent binding of calmodulin to neuronal gap junction proteins

We examined the interactions of calmodulin with neuronal gap junction proteins connexin35 (Cx35) from perch, its mouse homologue Cx36, and the related perch Cx34.7 using surface plasmon resonance. Calmodulin bound to the C-terminal domains of all three connexins with rapid kinetics in a concentration- and Ca2+-dependent manner. Dissociation was also very rapid. K(d)'s for calmodulin binding at a high-affinity site ranged from 11 to 72 nM, and K(1/2)'s for Ca2+ were between 3 and 5 microM. No binding to the intracellular loops was observed. Binding competition experiments with synthetic peptides mapped the calmodulin binding site to a 10-30 amino acid segment at the beginning of the C-terminal domain of Cx36. The micromolar K(1/2)'s and rapid on and off rates suggest that this interaction may change dynamically in neurons, and may occur transiently when Ca2+ is elevated to a level that would occur in the near vicinity of an activated synapse.     

Maturational alterations in gap junction expression and associated collagen synthesis in response to tendon function

Energy-storing tendons including the equine superficial digital flexor tendon (SDFT) contribute to energetic efficiency of locomotion at high-speed gaits, but consequently operate close to their physiological strain limits. Significant evidence of exercise-induced microdamage has been found in the SDFT which appears not to exhibit functional adaptation; the degenerative changes have not been repaired by the tendon fibroblasts (tenocytes), and are proposed to accumulate and predispose the tendon to rupture during normal athletic activity. The anatomically opposing common digital extensor tendon (CDET) functions only to position the digit, experiencing significantly lower levels of strain and is rarely damaged by exercise. A number of studies have indicated that tenocytes in the adult SDFT are less active in collagen synthesis and turnover than those in the immature SDFT or the CDET. Gap junction intercellular communication (GJIC) is known to be necessary for strain-induced collagen synthesis by tenocytes. We postulate therefore that expression of GJ proteins connexin 43 and 32 (Cx43; Cx32), GJIC and associated collagen expression levels are high in the SDFT and CDET of immature horses, when the SDFT in particular grows significantly in cross-sectional area, but reduce significantly during maturation in the energy-storing tendon only. The hypothesis was tested using tissue from the SDFT and CDET of foetuses, foals, and young adult Thoroughbred horses. Cellularity and the total area of both Cx43 and Cx32 plaques/mm² of tissue reduced significantly with maturation in each tendon. However, the total Cx43 plaque area per tenocyte significantly increased in the adult CDET. Evidence of recent collagen synthesis in the form of levels of neutral salt-soluble collagen, and collagen type I mRNA was significantly less in the adult compared with the immature SDFT; procollagen type I amino-propeptide (PINP) and procollagen type III amino-propeptide (PIIINP) levels per mm² of tissue and PINP expression per tenocyte also decreased with maturation in the SDFT. In the CDET PINP and PIIINP expression per tenocyte increased in the adult, and exceeded those in the adult SDFT. The level of PINP per mm² was greater in the adult CDET than in the SDFT despite the higher cellularity of the latter tendon. In the adult SDFT, levels of PIIINP were greater than those of PINP, suggesting relatively greater synthesis of a weaker form of collagen previously associated with microdamage. Tenocytes in monolayers showed differences in Cx43 and Cx32 expression compared with those in tissue, however there were age- and tendon-specific phenotypic differences, with a longer time for 50% recovery of fluorescence after photobleaching in adult SDFT cells compared with those from the CDET and immature SDFT. As cellularity reduces following growth in the SDFT, a failure of the remaining tenocytes to show a compensatory increase in GJ expression and collagen synthesis may explain why cell populations are not able to respond to exercise and to repair microdamage in some adult athletes. Enhancing GJIC in mature energy-storing tendons could provide a strategy to increase the cellular synthetic and reparative capacity.     

Complex formation between calmodulin and a peptide from the intracellular loop of the gap junction protein connexin43: Molecular conformation and energetics of binding

Gap junctions are formed by a family of transmembrane proteins, connexins. Connexin43 is a widely studied member of the family, being ubiquitously expressed in a variety of tissues and a target of a large number of disease mutations. The intracellular loop of connexin43 has been shown to include a calmodulin binding domain, but detailed 3-dimensional data on the structure of the complex are not available. In this study, we used a synthetic peptide from this domain to reveal the conformation of the calmodulin-peptide complex by small angle X-ray scattering. Upon peptide binding, calmodulin lost its dumbbell shape, adopting a more globular conformation. We also studied the energetics of the interaction using calorimetry and computational methods. All our data indicate that calmodulin binds to the peptide from cx43 in the classical ‘collapsed’ conformation.     

Magnesium gating of cardiac gap junction channels

We aimed to study kinetics of modulation by intracellular Mg²⁺ of cardiac gap junction (Mg²⁺ gate). Paired myocytes of guinea-pig ventricle were superfused with solutions containing various concentrations of Mg²⁺. In order to rapidly apply Mg²⁺ to one aspect of the gap junction, the non-junctional membrane of one of the pair was perforated at nearly the connecting site by pulses of nitrogen laser beam. The gap junction conductance (G_j) was measured by clamping the membrane potential of the other cell using two-electrode voltage clamp method. The laser perforation immediately increased G_j, followed by slow G_j change with time constant of 3.5 s at 10 mM Mg²⁺. Mg²⁺ more than 1.0 mM attenuated dose-dependently the gap junction conductance and lower Mg²⁺ (0.6 mM) increased G_j with a Hill coefficient of 3.4 and a half-maximum effective concentration of 0.6 mM. The time course of G_j changes was fitted by single exponential function, and the relationship between the reciprocal of time constant and Mg²⁺ concentration was almost linear. Based on the experimental data, a mathematical model of Mg²⁺ gate with one open state and three closed states well reproduced experimental results. One-dimensional cable model of thirty ventricular myocytes connected to the Mg²⁺ gate model suggested a pivotal role of the Mg²⁺ gate of gap junction under pathological conditions.     

Gap junction channels reconstituted in two closely apposed lipid bilayers

Intercellular communication mediated by gap junction channels plays an important role in many cellular processes. In contrast to other channels, gap junction channels span two plasma membranes resulting in an intracellular location for both ends of the junctional pore and the regulatory sites for channel gating. This configuration presents unique challenges for detailed experimental studies of junctional channel physiology and ligand-activation in situ. Availability of an appropriate model system would significantly facilitate future studies of gap junction channel function and structure. Here we show that the double-membrane channel can be reconstituted in pairs of closely apposed lipid bilayers, as experienced in cells. We have trapped the calcium-sensitive dye, arsenazo III (AIII), partially calcium-saturated (AIII-Ca), in one population of connexin32 reconstituted-liposomes, and EGTA in a second one. In such mixtures, the interaction of EGTA with AIII-Ca was measured by a large color shift from blue to red (decreased absorbance at 652 nm). The exchange of these compounds through gap junctions was proportional to these decrements. Results indicate that these connexon-mediated interliposomal channels are functional and are inhibited by the addition of alpha-glycyrrhetinic acid and by flufenamic acid, two gap junction communication inhibitors. Future use of this model system has the potential to improve our understanding of the permeability and modulation of junctional channels in its native intercellular 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.     

Specific Cx43 phosphorylation events regulate gap junction turnover in vivo

Gap junctions, composed of proteins from the connexin gene family, are highly dynamic structures that are regulated by kinase-mediated signaling pathways and interactions with other proteins. Phosphorylation of Connexin43 (Cx43) at different sites controls gap junction assembly, gap junction size and gap junction turnover. Here we present a model describing how Akt, mitogen activated protein kinase (MAPK) and src kinase coordinate to regulate rapid turnover of gap junctions. Specifically, Akt phosphorylates Cx43 at S373 eliminating interaction with zona occludens-1 (ZO-1) allowing gap junctions to enlarge. Then MAPK and src phosphorylate Cx43 to initiate turnover. We integrate published data with new data to test and refine this model. Finally, we propose that differential coordination of kinase activation and Cx43 phosphorylation controls the specific routes of disassembly, e.g., annular junction formation or gap junctions can potentially “unzip” and be internalized/endocytosed into the cell that produced each connexin.     

Expression of connexin 43 mRNA in microisolated murine osteoclasts and regulation of bone resorption in vitro by gap junction inhibitors

Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.     

Atrial fibrillation-associated Connexin40 mutants make hemichannels and synergistically form gap junction channels with novel properties

Mutations of Cx40 (GJA5) have been identified in people with lone chronic atrial fibrillation including G38D and M163V which were found in the same patient. We used dual whole cell patch clamp procedures to examine the transjunctional voltage (V_j) gating and channel conductance properties of these two rare mutants. Each mutant exhibited slight alterations of V_j gating properties and increased the gap junction channel conductance (γ_j) by 20–30 pS. While co-expression of the two mutations had similar effects on V_j gating, it synergistically increased γ_j by 50%. Unlike WTCx40 or M163V, G38D induced activity of a dominant 271 pS hemichannel.     

Granulocyte colony-stimulating factor activates Wnt signal to sustain gap junction function through recruitment of beta-catenin and cadherin

Our previous study reveals that connexin (Cx) 43 is targeted by ACh to prevent lethal arrhythmia. Granulocyte colony-stimulating factor (G-CSF), used against ischemic heart failure, may be another candidate, however, with unknown mechanisms. Therefore, we investigated the cellular effects of G-CSF. G-CSF activated the Wnt and Jak2 signals in cardiomyocytes, and up-regulated Cx43 protein and phosphorylation levels. In addition, G-CSF enhanced the localization of Cx43, beta-catenin and cadherin on the plasma membrane. G-CSF inhibited the reduction of Cx43 by enhancing Cx43 anchoring and sustained the cell-cell communication during hypoxia. Consequently, G-CSF suppressed ventricular arrhythmia induced by myocardial infarction. As a result, G-CSF could be used as a therapeutic tool for arrhythmia.     

Degradation of connexins and gap junctions

Connexin proteins are short-lived within the cell, whether present in the secretory pathway or in gap junction plaques. Their levels can be modulated by their rate of degradation. Connexins, at different stages of assembly, are degraded through the proteasomal, endo-/lysosomal, and phago-/lysosomal pathways. In this review, we summarize the current knowledge about connexin and gap junction degradation including the signals and protein–protein interactions that participate in their targeting for degradation.