Intercellular Gap Junction Channels Research Articles

Connexins in Lens Development and Cataractogenesis

The lens is an avascular organ that transmits and focuses light images onto the retina. Intercellular gap junction channels, formed by at least three different connexin protein subunits, alpha1 (connexin43 or Gja1), alpha3 (connexin46 or Gja3) and alpha8 (connexin50 or Gja8), are utilized to transport metabolites, ions and water in the lens. In combination with physiological and biochemical analyses, recent genetic studies have significantly improved our understanding about the roles of diverse gap junction channels formed by alpha3 and alpha8 connexin subunits during lens development and cataract formation. These studies have demonstrated that alpha3 connexin is essential for lens transparency while alpha8 connexin is important for lens growth and transparency. Diverse gap junction channels formed by alpha3 and alpha8 subunits are important for the differentiation, elongation and maturation of lens fiber cells. Aberrant gap junction communication, caused by alterations of channel assembly, channel gating or channel conductance, can lead to different types of cataracts. These findings provide some molecular insights for essential roles of connexins and gap junctions in lens formation and the establishment and maintenance of lifelong lens transparency.

An Innexin-Dependent Cell Network Establishes Left-Right Neuronal Asymmetry in C. elegans

Gap junctions are widespread in immature neuronal circuits, but their functional significance is poorly understood. We show here that a transient network formed by the innexin gap-junction protein NSY-5 coordinates left-right asymmetry in the developing nervous system of Caenorhabditis elegans. nsy-5 is required for the left and right AWC olfactory neurons to establish stochastic, asymmetric patterns of gene expression during embryogenesis. nsy-5-dependent gap junctions in the embryo transiently connect the AWC cell bodies with those of numerous other neurons. Both AWCs and several other classes of nsy-5-expressing neurons participate in signaling that coordinates left-right AWC asymmetry. The right AWC can respond to nsy-5 directly, but the left AWC requires nsy-5 function in multiple cells of the network. NSY-5 forms hemichannels and intercellular gap-junction channels in Xenopus oocytes, consistent with a combination of cell-intrinsic and network functions. These results provide insight into gap-junction activity in developing circuits.

Connexin expression and gap junctional intercellular communication in human squamous cell carcinoma of the head and neck

Our laboratory is investigating the role that gap junction intercellular channels (composed of proteins called connexins) play in communicating apoptotic signals from therapeutically targeted squamous cell carcinoma of the head and neck (SCCHN) cells to adjacent, untreated, "bystander" cells (bystander effect). The nature of this research underscores the importance of delineating connexin expression patterns in SCCHN, and how this correlates with gap junctional intercellular communication (GJIC) and bystander effects. The GJIC activity of a diverse panel of SCCHN cell lines, as well as normal oral epithelial (NOE) cell controls was determined in vitro. These data were correlated with connexin expression patterns determined through connexin 43 and connexin 26 immunofluorescence. Cell lines with retained GJIC activity all expressed connexin 43 on the cell membrane. Cell lines that did not communicate microinjected lucifer yellow (lost GJIC activity) showed no connexin expression, either at the cell membrane or in the cytosol. Connexin 26 was not expressed in any of our SCCHN cell lines, whereas both connexin 43 and connexin 26 were expressed in the NOE cell controls. Furthermore, connexin 43 introduction into a GJIC (and connexin) deficient SCCHN cell line conferred no growth inhibitory effect. Connexin 43 expression correlates with retained GJIC in SCCHN in vitro. Connexin 26 may have a role as a tumor suppressor in SCCHN. The data presented have relevance to our ongoing investigations of gap-junction mediated bystander effects in SCCHN and are being expanded to investigations on actual SCCHN tumor specimens.

The gap junction cellular internet: connexin hemichannels enter the signalling limelight.

Cxs (connexins), the protein subunits forming gap junction intercellular communication channels, are transported to the plasma membrane after oligomerizing into hexameric assemblies called connexin hemichannels (CxHcs) or connexons, which dock head-to-head with partner hexameric channels positioned on neighbouring cells. The double membrane channel or gap junction generated directly couples the cytoplasms of interacting cells and underpins the integration and co-ordination of cellular metabolism, signalling and functions, such as secretion or contraction in cell assemblies. In contrast, CxHcs prior to forming gap junctions provide a pathway for the release from cells of ATP, glutamate, NAD+ and prostaglandin E2, which act as paracrine messengers. ATP activates purinergic receptors on neighbouring cells and forms the basis of intercellular Ca2+ signal propagation, complementing that occuring more directly via gap junctions. CxHcs open in response to various types of external changes, including mechanical, shear, ionic and ischaemic stress. In addition, CxHcs are influenced by intracellular signals, such as membrane potential, phosphorylation and redox status, which translate external stresses to CxHc responses. Also, recent studies demonstrate that cytoplasmic Ca2+ changes in the physiological range act to trigger CxHc opening, indicating their involvement under normal non-pathological conditions. CxHcs not only respond to cytoplasmic Ca2+, but also determine cytoplasmic Ca2+, as they are large conductance channels, suggesting a prominent role in cellular Ca2+ homoeostasis and signalling. The functions of gap-junction channels and CxHcs have been difficult to separate, but synthetic peptides that mimic short sequences in the Cx subunit are emerging as promising tools to determine the role of CxHcs in physiology and pathology.

Absence of α3 (Cx46) and α8 (Cx50) connexins leads to cataracts by affecting lens inner fiber cells

Lens development and transparency have been hypothesized to depend on intercellular gap junction channels, consisting of α3 (Cx46) and α8 (Cx50) connexin subunits, to transport metabolites, secondary messages and ions between lens cells. To evaluate this hypothesis, we have generated α3(−/−) α8(−/−) double knockout mice and characterized their lens phenotypes. Without gap junctions between lens fiber cells, α3(−/−) α8(−/−) lenses displayed severe cataracts resulting from cell swelling and degeneration of inner fibers while normal peripheral fiber cells continued to form throughout life. Neither an increase of degraded crystallins nor an increase of water-insoluble crystallins was found in α3(−/−) α8(−/−) lenses. However, a substantial reduction of γ-crystallin proteins, but not α- and β-crystallins, was detected. These results suggest that gap junction communication is important for maintaining lens homeostasis of inner fiber cells and that a loss of gap junctions leads to cataract formation as well as reductions of γ-crystallin proteins and transcripts.

Single-cell microinjection of cytochromec can result in gap junction-mediated apoptotic cell death of bystander cells in head and neck cancer

Gap junction intercellular channels are required for metabolic cooperation between cells and regulate normal tissue homeostasis by means of the transfer of small molecules between contacting cells. Not surprisingly, the gap junction phenotype is frequently lost during carcinogenesis in human tissues (including those of the upper aerodigestive tract), freeing individual cancer cells from the growth control signals of normal surrounding tissues and less aggressive adjacent cancer cells. We hypothesized that gap junctional intercellular communication (GJIC) could mediate a bystander effect (apoptotic cell death) in squamous cell carcinoma of the head and neck (SCCHN) cells adjacent to individually targeted SCCHN cells. Single-cell microinjection of cytochrome c was used to induce apoptosis in target SCCHN cells with endogenous GJIC activity and in an SCCHN cell line with exogenously introduced GJIC activity. Apoptosis was followed in target and surrounding bystander cells through light and time course microscopic characterization. All of the preceding experiments were carried out in the absence and presence of 18-beta-glycerretinic acid, a pharmacologic inhibitor of GJIC. When cytochrome c was introduced into SCCHN cells with endogenous GJIC activity through single-cell microinjection, bystander effects (apoptosis of nontarget cells) were observed. When GJIC activity was blocked with the specific pharmacologic inhibitor of gap junctions, 18-beta-glycerretinic acid, a bystander effect was never seen in GJIC active SCCHN cell lines. Gap junction intercellular channels can mediate a bystander effect in SCCHN. Inconsistencies in our data will be discussed in the context of recent advances in this field, as well as our future research directions.

A C α Model for the Transmembrane α Helices of Gap Junction Intercellular Channels

Gap junction channels connect the cytoplasms of apposed cells via an intercellular conduit formed by the end-to-end docking of two hexameric hemichannels called connexons. We used electron cryomicroscopy to derive a three-dimensional density map at 5.7 Å in-plane and 19.8 Å vertical resolution, allowing us to identify the positions and tilt angles for the 24 α helices within each hemichannel. The four hydrophobic segments in connexin sequences were assigned to the α helices in the map based on biochemical and phylogenetic data. Analyses of evolutionary conservation and compensatory mutations in connexin evolution identified the packing interfaces between the helices. The final model, which specifies the coordinates of C α atoms in the transmembrane domain, provides a structural basis for understanding the different physiological effects of almost 30 mutations and polymorphisms in terms of structural deformations at the interfaces between helices, revealing an intimate connection between molecular structure and disease.

Histamine inhibits conducted vasodilation through endothelium-derived NO production in arterioles of mouse skeletal muscle.

Conducted vasodilation along arterioles manifests the spread of hyperpolarization through gap junction channels along endothelium. Whereas histamine increases the permeability of capillary and venular endothelium, its effect on the integrity of arteriolar endothelium is unknown. We tested whether histamine could inhibit conducted vasodilation. In second-order arterioles (2A) supplying the cremaster muscle of C57BL6, PECAM-1-/-, and eNOS-/- mice (8-12 wk), neither resting (16+/-2 microm) nor maximal (38+/-2 microm) diameters were different. Acetylcholine (ACh) microiontophoresis (1 microA, 500 ms pulse) triggered vasodilation that was conducted >1400 microm along arterioles. Neither local (14+/-2 microm) nor conducted vasodilation (10+/-2 microm) was different among mice. Histamine (5 microM) had no effect on resting diameter or local vasodilation to ACh yet inhibited conduction by >50% in C57BL6 and PECAM-1-/- mice (P<0.05); this effect was abolished after blockade of NO synthase or of soluble guanylate cyclase. Washout of histamine restored conduction, though recovery was longer (P<0.05) in PECAM-1-/- mice vs. C57BL6 mice. Remarkably, conducted vasodilation in eNOS-/- mice was insensitive to histamine. These findings indicate that histamine inhibits cell-to-cell coupling through an NO-dependent mechanism and suggests a dynamic interaction among intercellular adhesion molecules and gap junction channels along arteriolar endothelium.

Connexin 40 expression in bovine and rat retinas.

Retinal neurons are extensively coupled through gap junction intercellular channels, but few connexin subtypes have been identified in mammalian retinal neurons. Based on previous findings that retinal gap junctional coupling is modulated by both dopamine and nitric oxide, presumably through connexin phosphorylation, we examined whether the connexin phosphoprotein subtype, connexin 40 (Cx40), was expressed in mammalian retinas. Immunostaining of rat and bovine retinas using Cx40-specific antibodies from two independent sources showed punctate staining between cells in the outer nuclear layer (ONL) and a sublayer of cells within the inner nuclear layer (INL). In addition, sparse punctate staining was detected in the ganglion cell/axon fiber layers (GCL/AFL). No punctate staining was observed in the outer (OS) or inner segment (IS) layers, and rarely in the outer plexiform layer (OPL) or inner plexiform layer (IPL). Double immunostaining of bovine retinas with antibodies to G(o), which stains bipolar cells, and to Cx40, showed little overlap, suggesting these bipolar cells do not express Cx40. Western blot analysis of alkaline-extracted bovine retinal membranes revealed Cx40 immunopositive bands of about 40 kD (monomer) and 80 kD (dimer). In both locations (monomer and dimer), the bands appeared as doublets, and their immunoreactivity was abolished when the antibody was pre-adsorbed with immunogenic Cx40 peptide. The doublet at 40 kD co-migrated with an immunopositive doublet present in heart membranes. Treatment with alkaline phosphatase altered the banding pattern of Cx40. The results suggest that the connexin phosphoprotein subtype, Cx40, is expressed within the neural layers of the mammalian retina.

Evidence of Gap Junctions in the Stable Nonobstructed Human Bladder

Evidence of Gap Junctions in the Stable Nonobstructed Human Bladder

Gap junctions and connexins: potential contributors to the immunological synapse

Gap junctional communication is a widespread mechanism for metabolic coupling of adjoining cells. In the immune system, evidence has built up showing that lymphocytes possess the protein building blocks of gap junctions, the connexins. The most widespread is connexin 43, but connexin 40 is also present in secondary lymphoid organs. Inhibitors of gap junctional communication, especially the highly specific connexin mimetic peptides, have been shown to decrease the secretion of immunoglobulins and cytokines by T and B lymphocyte cocultures, indicating that connexins may play a fundamental role in lymphocyte physiology. Traditionally, connexins function when assembled into gap junction-intercellular channels. However, the possibility is now arising that gap junction hemichannels, previously viewed as plasma membrane precursors of gap junctions, are also involved in the release from cells of small metabolites, e.g., adenosine 5'-triphosphate and nicotinamide adenine dinucleotide(+), and this opens up a second, possible paracrine function for connexins detected in lymphocytes. The increasing structural and functional evidence points to a potential role that lymphocyte gap junctional intercellular communication may play within the complex signaling components of the immunological synapse.

Lighting up gap junction channels in a flash.

Gap junction intercellular communication channels permit the exchange of small regulatory molecules and ions between neighbouring cells and coordinate cellular activity in diverse tissue and organ systems. These channels have short half-lives and complex assembly and degradation pathways. Much of the recent work elucidating gap junction biogenesis has featured the use of connexins (Cx), the constituent proteins of gap junctions, tagged with reporter proteins such as Green Fluorescent Protein (GFP) and has illuminated the dynamics of channel assembly in live cells by high-resolution time-lapse microscopy. With some studies, however, there are potential short-comings associated with the GFP chimeric protein technologies. A recent report by Gaietta et al., has highlighted the use of recombinant proteins with tetracysteine tags attached to the carboxyl terminus of Cx43, which differentially labels 'old' and 'new' connexins thus opening up new avenues for studying temporal and spatial localisation of proteins and in situ trafficking events.

Gap junction intercellular communication during lymphocyte transendothelial migration.

Migration of lymphocytes across the endothelium of central or peripheral tissues, a process occurring following activation or differentiation, involves cell to cell interactions featuring adhesion and heterotypic signalling 'cross-talk'. Since lymphocytes and endothelial cells express connexins, the subunit proteins of gap junction intercellular channels, we investigated whether these channels feature in heterotypic signalling during transendothelial migration of lymphocytes. We show, using FACS analysis, that calcein, a gap junction permeant fluorescent dye, was transferred from endothelial cell layers to lymphocytes. The gap junction involvement in intercellular dye transfer was reinforced by studies showing that the process was inhibited by connexin mimetic peptides, a new class of reagents shown to block gap junction communication. Further evidence for the involvement of lymphocyte gap junctions in intercellular communication during transendothelial migration was obtained by two-photon laser scanning microscopy. Although gap junctional communication was inhibited by connexin mimetic peptides, they had little influence on the transmigration process.

Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels.

The assembly of gap junctions was investigated in mammalian cells expressing connexin (Cx) 26, 32 and 43 fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Targeting of Cx32-CFP and 43-GFP to gap junctions and gap junctional communication was inhibited in cells treated with Brefeldin A, a drug that disassembles the Golgi. However gap junctions constructed of Cx26-GFP were only minimally affected by Brefeldin A. Nocodazole, a microtubule disruptor, had little effect on the assembly of Cx43-GFP gap junctions, but perturbed assembly of Cx26-GFP gap junctions. Co-expression of Cx26-YFP and Cx32-CFP in cells treated with Brefeldin A resulted in assembly of gap junctions constructed of Cx26-YFP. Two amino acids that distinguish Cx26 from Cx32 in transmembrane domains were mutated in Cx32 to investigate underlying mechanisms determining trafficking routes to gap junctions. One mutation, Cx32I28L, conferred on it partial Cx26-like trafficking properties as well the post-translational membrane insertion characteristics of Cx26, suggesting that a key determinant regulating trafficking was present in the first transmembrane domain. The results provide a protein trafficking basis for specifying and regulating connexin composition of gap junctions and thus selectivity of intercellular signaling, with Cx32 and 43 trafficking through the secretory pathway and Cx26 also following an alternative pathway.

Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants.

The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.

Cardiac gap junction channels: modulation of expression and channel properties.

In the heart, intercellular gap junction channels constructed from connexin molecules are crucial for conduction of the electric impulse. Cardiomyocytes can be interconnected by channels composed of three types of connexin proteins: Cx40, Cx43 or Cx45. In mammalian hearts, these three isoforms are regionally differently expressed and even between the species differences exist. Each of these channel-types possesses specific properties and are susceptible to modulation by various mechanisms. In this paper we compare the differences in properties of these channels as deduced from studies on transfected cells and isolated cardiomyocytes and discuss the factors involved in modulation of channel properties. Next, we evaluate the consequences of alterations in expression and modulation of channel properties for cardiac function. Therefore, we have compared reports on genetically engineered animals and discuss this information in relation to various pathophysiological disorders.

Immunoglobulin and cytokine expression in mixed lymphocyte cultures is reduced by disruption of gap junction intercellular communication.

Connexins (Cx), the protein subunits assembled into gap junction intercellular communication channels, are expressed in primary lymphoid organs and by circulating leukocytes. Human tonsil-derived T and B lymphocytes express Cx40 and 43; circulating human T, B, and NK lymphocytes express Cx43 and directly transfer between each other a low molecular dye indicative that functional gap junctions exist. We now identify specific properties in the immune system underwritten by gap junctions. Mixed lymphocytes cultured in the presence of two reagents with independent inhibitory action on gap junction communication, a connexin mimetic peptide and 18-alpha-glycyrrhetinic acid, markedly reduced the secretion of IgM, IgG, and IgA. The secretion of these immunoglobulins by purified B cells was also reduced by the two classes of gap junction inhibitors. Complex temporal inhibitory effects on the expression of mRNA encoding interleukins, especially IL-10, were also observed. The results indicate that intercellular signaling across gap junctions is an important component of the mechanisms underlying metabolic cooperation in the immune system.

Application of SCAM (substituted cysteine accessibility method) to gap junction intercellular channels.

The pore-lining residues of gap junction channels determine their permeability to ions and small cellular metabolites. These residues can be identified through systematic cysteine substitution and accessibility analysis, commonly known as SCAM (Substituted Cysteine Accessibility Method). However, application of this technique to intercellular channels is more complicated than for their transmembrane counterparts. We have utilized a novel dual-oocyte perfusion device to apply cysteine reagents to the cytoplasmic face of paired, voltage-clamped Xenopus oocytes. In this configuration, a large and irreversible cysteine reagent MBB (maliemidobutyryl biocytin, mw 537) was shown to readily traverse the gap junction pore and induce conductance changes upon reaction of accessible sites. Of the 11 reactive sites identified, 6 were located in M3, where they span the bilayer. They display a periodicity characteristic of the tilted helix that lines the pore in the gap junction structure of Unger et al. (1999). Access to several of the other sites was attributed to aqueous crevices between transmembrane helices. Reactive sites were slightly different than those identified for gap junction hemichannels (Zhou et al. 1997), suggesting that conformational changes occur upon docking.

Unique and shared functions of different connexins in mice.

Connexins are the protein subunits of intercellular gap junction channels. In mammals, they are encoded by a family of at least 15 genes, which show cell-type-specific but overlapping patterns of expression. Mice lacking connexin43 (Cx43) die postnatally from obstruction of the right ventricular outflow tract of the heart. To discriminate between the unique and shared functions of Cx43, Cx40 and Cx32, we generated two 'knock-in' mouse lines, Cx43KI32 and Cx43KI40, in which the coding region of the Cx43 gene was replaced, respectively, by the coding regions of Cx32 or Cx40. Heterozygous mutants were fertile and co-expressed the wild-type and the corresponding recombinant allele in all tissues analyzed. Heterozygous Cx43KI32, but not Cx43KI40, mutant mothers were unable to nourish their pups to weaning age, possibly reflecting a defect in milk ejection. Homozygous mutant males were sterile because of extensive germ-cell deficiency. The ovaries of homozygous Cx43KI32 neonates exhibited all stages of follicular development and ovulation. The hearts of homozygous Cx43KI32 neonates showed mild morphological defects, but the cardiac morphology of homozygous Cx43KI40 neonates was relatively normal. Spontaneous ventricular arrhythmias were observed in most Cx43KI40 and some Cx43KI32 mutant mice, suggesting increased ventricular vulnerability in these mice. The postnatal lethality of Cx43-deficient mice was rescued in Cx43KI32 or Cx43KI40 mice, indicating that Cx43, Cx40 and Cx32 share at least some vital functions. On the other hand, Cx43KI32 and Cx43KI40 mice differed functionally and morphologically from each other and from wild-type mice. Thus, these connexins also have unique functions.

Biosynthesis and structural composition of gap junction intercellular membrane channels

Gap junction channels assemble as dodecameric complexes, in which a hexameric connexon (hemichannel) in one plasma membrane docks end-to-end with a connexon in the membrane of a closely apposed cell to provide direct cell-to-cell communication. Synthesis, assembly, and trafficking of the gap junction channel subunit proteins referred to as connexins, largely appear to follow the general secretory pathway for membrane proteins. The connexin subunits can assemble into homo-, as well as distinct hetero-oligomeric connexons. Assembly appears to be based on specific signals located within the connexin polypeptides. Plaque formation by the clustering of gap junction channels in the plane of the membrane, as well as channel degradation are poorly understood processes that are topics of current research. Recently, we tagged connexins with the autofluorescent reporter green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants and combined this reporter technology with single, and dual-color, high resolution deconvolution microscopy, computational volume rendering, and time-lapse microscopy to examine the detailed organization, structural composition, and dynamics of gap junctions in live cells. This technology provided for the first time a realistic, three-dimensional impression of gap junctions as they appear in the plasma membranes of adjoining cells, and revealed an excitingly detailed structural organization of gap junctions never seen before in live cells. Here, I summarize recent progress in areas encompassing the synthesis, assembly and structural composition of gap junctions with a special emphasis on the recent results we obtained using cell-free translation/ membrane-protein translocation, and autofluorescent reporters in combination with live-cell deconvolution microscopy.