Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions.

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.     

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.     

Extracellular K+ and Astrocyte Signaling via Connexin and Pannexin Channels

Astrocytes utilize two major pathways to achieve long distance intercellular communication. One pathway involves direct gap junction mediated signal transmission and the other consists of release of ATP through pannexin channels and excitation of purinergic receptors on nearby cells. Elevated extracellular potassium to levels occurring around hyperactive neurons affects both gap junction and pannexin1 channels. The action on Cx43 gap junctions is to increase intercellular coupling for a period that long outlasts the stimulus. This long term increase in coupling, termed “LINC”, is mediated through calcium and calmodulin dependent activation of calmodulin dependent kinase (CaMK). Pannexin1 can be activated by elevations in extracellular potassium through a mechanism that is quite different. In this case, potassium shifts activation potentials to more physiological range, thereby allowing channel opening at resting or slightly depolarized potentials. Enhanced activity of both these channel types by elevations in extracellular potassium of the magnitude occurring during periods of high neuronal activity likely has profound effects on intercellular signaling among astrocytes in the nervous system.     

SRC utilizes Cas to block gap junctional communication mediated by connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.     

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.     

Modulation of human fibroblast gap junction intercellular communication by hyaluronan

The composition of the extracellular matrix changes during dermal repair. Initially, hyaluronan (HA) concentration is high, however, by day 3, HA is eliminated. HA optimizes collagen organization within granulation tissue. One possible mechanism of HA modulation of collagen packing is through the promotion of gap junction intercellular communication (GJIC). Gap junctions are gated channels that allow rapid intercellular communication and synchronization of coupled cell activities. The gap junction channel is composed of connexin (Cx) proteins that form a gated channel between coupled cells. HA is reported to enhance Cx43 expression in transformed fibroblasts. GJIC was quantified by the scrape loading technique and reported as a coupling index. The coupling index for human dermal fibroblasts was 4.6 +/- 0.2, while the coupling index for fibroblasts treated with HA more than doubled to 10.6 +/- 0.7. By Western blot analysis no differences were appreciated in the protein levels of Cx43 or beta-catenin, a protein involved in the translocation of Cx to the cell surface. By immuno-histology Cx43 and beta-catenin were evenly distributed throughout the cell in controls, but in cells treated with HA these proteins were co-localized to the cell surface. Coupled fibroblasts are reported to enhance the organization of collagen fibrils. It is proposed that HA increases the accumulation of Cx43 and beta-catenin on the cell surface, leading to greater GJIC and enhanced collagen organization.     

Sharing signals: connecting lung epithelial cells with gap junction channels

Gap junction channels enable the direct flow of signaling molecules and metabolites between cells. Alveolar epithelial cells show great variability in the expression of gap junction proteins (connexins) as a function of cell phenotype and cell state. Differential connexin expression and control by alveolar epithelial cells have the potential to enable these cells to regulate the extent of intercellular coupling in response to cell stress and to regulate surfactant secretion. However, defining the precise signals transmitted through gap junction channels and the cross talk between gap junctions and other signaling pathways has proven difficult. Insights from what is known about roles for gap junctions in other systems in the context of the connexin expression pattern by lung cells can be used to predict potential roles for gap junctional communication between alveolar epithelial cells.     

Gap junction channel gating modulated through protein phosphorylation

As a ubiquitous post-translation modification process, protein phosphorylation has proven to be a key mechanism in regulating the function of several membrane proteins, including transporters and channels. Connexins, pannexins, and innexins are protein families that form gap junction channels essential for intercellular communication. Connexins have been intensely studied, and most of their isoforms are known to be phosphorylated by protein kinases that lead to modifications in tyrosine, serine, and threonine residues, which have been reported to affect, in one way or another, intercellular communication. Despite the abundant reports on changes in intercellular communication due to the activation or inactivation of numerous kinases, the molecular mechanisms by which phosphorylation alters channel gating properties have not been elucidated completely. Hence, this chapter will cover some of the current, relevant research that attempt to explain how phosphorylation triggers and/or modulates gap junction channel gating.     

Communication compartments in mixed cell cultures

Mixed cultures of epithelial (BRL) cells and fibroblasts (BHK), which sort themselves out into separate domains of each cell type, form communication compartments. Electrical coupling, dye coupling and metabolic coupling measurements have been used to show that small ions and molecules can move freely via intercellular junctions between all the cells in a domain, while their movement across the boundaries between domains is severely restricted. Metabolic coupling is the most sensitive method for detecting trans-boundary communication but the results obtained from all three methods are compatible. The data suggest the reduced transfer across the boundaries is due to fewer channels, resulting from a lower frequency of junction formation between heterologous cells, rather than to channels of smaller diameter. Concentration gradients of small cytoplasmic molecules can be established within these communication compartments which are similar to those predicted to explain pattern formation in developing systems. It is suggested that the cell surface features which cause this sorting out are also responsible for the reduced frequency of heterologous junction formation and hence for compartmentalization.     

Pharmacology of gap junctions. New pharmacological targets for treatment of arrhythmia, seizure and cancer?

Intercellular communication in many organs is maintained via intercellular gap junction channels composed of connexins, a large protein family with a number of isoforms. This gap junction intercellular communication (GJIC) allows the propagation of action potentials (e.g., in brain, heart), and the transfer of small molecules which may regulate cell growth, differentiation and function. The latter has been shown to be involved in cancer growth: reduced GJIC often is associated with increased tumor growth or with de-differentiation processes. Disturbances of GJIC in the heart can cause arrhythmia, while in brain electrical activity during seizures seems to be propagated via gap junction channels. Many diseases or pathophysiological conditions seem to be associated with alterations of gap junction protein expression. Thus, depending on the target disease opening or closure of gap junctions may be of interest, or alteration of connexin expression. GJIC can be affected acutely by changing gap junction conductance or--more chronic--by altering connexin expression and membrane localisation. This review gives an overview on drugs affecting GJIC.     

Calcium and connexin-based intercellular communication, a deadly catch?

Ca²⁺ is known as a universal messenger mediating a wide variety of cellular processes, including cell death. In fact, this ion has been proposed as the ‘cell death master’, not only at the intracellular but also at the intercellular level. The most direct form of intercellular spread of cell death is mediated by gap junction channels. These channels have been shown to propagate cell death as well as cell survival signals between the cytoplasm of neighbouring cells, reflecting the dual role of Ca²⁺ signals, i.e. cell death versus survival. Its precursor, the unopposed hemichannel (half of a gap junction channel), has recently joined in as a toxic pore connecting the intracellular with the extracellular environment and allowing the passage of a range of substances. The biochemical nature of the so-called intercellular cell death molecule, transferred through gap junctions or released/taken up via hemichannels, remains elusive but several studies pinpoint Ca²⁺ itself or its messenger inositol trisphosphate as the responsible masters in crime. Although direct evidence is still lacking, indirect data including Ca²⁺ involvement in intercellular communication and cell death, and effects of intercellular communication on intracellular Ca²⁺ homeostasis, support this hypothesis. In addition, hemichannels and their molecular building blocks, connexin or pannexin proteins, may exert their effects on Ca²⁺-dependent cell death at the intracellular level, independently from their channel functions. This review provides a cutting edge overview of the current knowledge and underscores the intimate connection between intercellular communication, Ca²⁺ signalling and cell death.     

Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia

Gap junctions serve as intercellular conduits that allow the exchange of small molecular weight molecules (up to 1 kDa) including ions, metabolic precursors and second messengers. Microglia are capable of recognizing peptidoglycan (PGN) derived from the outer cell wall of Staphylococcus aureus, a prevalent CNS pathogen, and respond with the robust elaboration of numerous pro-inflammatory mediators. Based on recent reports demonstrating the ability of tumor necrosis factor-alpha and interferon-gamma to induce gap junction coupling in macrophages and microglia, it is possible that pro-inflammatory mediators released from PGN-activated microglia are capable of inducing microglial gap junction communication. In this study, we examined the effects of S. aureus-derived PGN on Cx43, the major connexin in microglial gap junction channels, and functional gap junction communication using single-cell microinjections of Lucifer yellow (LY). Exposure of primary mouse microglia to PGN led to a significant increase in Cx43 mRNA and protein expression. LY microinjection studies revealed that PGN-treated microglia were functionally coupled via gap junctions, the specificity of which was confirmed by the reversal of activation-induced dye coupling by the gap junction blocker 18-alpha-glycyrrhetinic acid. In contrast to PGN-activated microglia, unstimulated cells consistently failed to exhibit LY dye coupling. These results indicate that PGN stimulation can induce the formation of a functional microglial syncytium, suggesting that these cells may be capable of influencing neuro-inflammatory responses in the context of CNS bacterial infections through gap junction intercellular communication.     

Roles and regulation of lens epithelial cell connexins

The avascular lens of the eye is covered anteriorly by an epithelium containing nucleated, metabolically active cells. This epithelium contains the first lens cells to encounter noxious external stimuli and cells that can develop compensatory or protective responses. Lens epithelial cells express the gap junction proteins, connexin43 (Cx43) and connexin50 (Cx50). Cx43 and Cx50 form gap junction channels and hemichannels with different properties. Although they may form heteromeric hemichannels, Cx43 and Cx50 probably do not form heterotypic channels in the lens. Cx50 channels make their greatest contribution to intercellular communication during the early postnatal period; subsequently, Cx43 becomes the predominant connexin supporting intercellular communication. Although epithelial Cx43 appears dispensable for lens development, Cx50 is critical for epithelial cell proliferation and differentiation. Cx43 and Cx50 hemichannels and gap junction channels are regulated by multiple different agents. Lens epithelial cell connexins contribute to both normal lens physiology and pathology.     

Connexin and pannexin mediated cell-cell communication

In this review, we briefly summarize what is known about the properties of the three families of gap junction proteins, connexins, innexins and pannexins, emphasizing their importance as intercellular channels that provide ionic and metabolic coupling and as non-junctional channels that can function as a paracrine signaling pathway. We discuss that two distinct groups of proteins form gap junctions in deuterostomes (connexins) and protostomes (innexins), and that channels formed of the deuterostome homologues of innexins (pannexins) differ from connexin channels in terms of important structural features and activation properties. These differences indicate that the two families of gap junction proteins serve distinct, complementary functions in deuterostomes. In several tissues, including the CNS, both connexins and pannexins are involved in intercellular communication, but have different roles. Connexins mainly contribute by forming the intercellular gap junction channels, which provide for junctional coupling and define the communication compartments in the CNS. We also provide new data supporting the concept that pannexins form the non-junctional channels that play paracrine roles by releasing ATP and, thus, modulating the range of the intercellular Ca(2+)-wave transmission between astrocytes in culture.     

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.     

Modulation of junctional permeability

Changes in intercellular coupling can be accomplished by continuous synthesis and destruction of intercellular channels and through a modulation of unit channel permeability. The increase in free [Ca2+]i caused by activation of sodium-calcium exchange or by metabolic inhibition leads to cell decoupling. In embryonic cells the conductance of the gap junction is strongly dependent on pHi. The exact role of Ca2+ and H+ in the physiological modulation of junctional conductance remains unknown. The cyclic AMP (cAMP)-calcium hypothesis is presented. According to this view, cAMP modulates the junctional permeability through specific kinases. A feedback mechanism between calcium and cAMP might be relevant in the physiological control of junctional conductance.     

Quantitative analysis of gap-junctional intercellular communication in precision-cut mouse liver slices

Direct intercellular communication through gap junction channels is involved in the maintenance of tissue homeostasis and suppression of carcinogenesis. Gap-junctional communication is often altered in tumor cells but it can also be modulated in response to tumor promotors or inflammatory signals. In order to evaluate the effect of nongenotoxic compounds, suggested to be involved in tumor promotion, on gap junctional intercellular communication in the liver, we have developed a direct dye transfer method. The fluorescent dye Alexa Fluor 488 was iontophoretically injected into hepatocytes of freshly prepared, precision-cut mouse liver slices (250 microm). The area of dye spreading was monitored and quantified by microscopy. Comparison of dye spreading in connexin-32-deficient versus wild-type liver revealed a 96% decrease in connexin-32-deficient tissue. Induction of an acute phase response in connexin-32-deficient mice by intraperitoneal injection of lipopolysaccharide increased dye coupling by 33%, probably due to upregulation of connexin-26-containing gap junction channels.     

Gap junction channels exhibit connexin-specific permeability to cyclic nucleotides

Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (I(h)) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in I(h) current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K(+) permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were approximately 10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 > or = Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell.