Cell communication across gap junctions: a historical perspective and current developments.

Collaborative communication lies at the centre of multicellular life. Gap junctions (GJs) are surface membrane structures that allow direct communication between cells. They were discovered in the 1960s following the convergence of the detection of low-resistance electrical interactions between cells and anatomical studies of intercellular contact points. GJs purified from liver plasma membranes contained a 27 kDa protein constituent; it was later named Cx32 (connexin 32) after its full sequence was determined by recombinant technology. Identification of Cx43 in heart and later by a further GJ protein, Cx26 followed. Cxs have a tetraspan organization in the membrane and oligomerize during intracellular transit to the plasma membrane; these were shown to be hexameric hemichannels (connexons) that could interact end-to-end to generate GJs at areas of cell-to-cell contact. The structure of the GJ was confirmed and refined by a combination of biochemical and structural approaches. Progress continues towards obtaining higher atomic 3D resolution of the GJ channel. Today, there are 20 and 21 highly conserved members of the Cx family in the human and mouse genomes respectively. Model organisms such as Xenopus oocytes and zebra fish are increasingly used to relate structure to function. Proteins that form similar large pore membrane channels in cells called pannexins have also been identified in chordates. Innexins form GJs in prechordates; these two other proteins, although functionally similar, are very different in amino acid sequence to the Cxs. A time line tracing the historical progression of wide ranging research in GJ biology over 60 years is mapped out. The molecular basis of channel dysfunctions in disease is becoming evident and progress towards addressing Cx channel-dependent pathologies, especially in ischaemia and tissue repair, continues.

Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury.

Connexin-43 (Cx43), a predominant cardiac connexin, forms gap junctions (GJs) that facilitate electrical cell-cell coupling and unapposed/nonjunctional hemichannels that provide a pathway for the exchange of ions and metabolites between cytoplasm and extracellular milieu. Uncontrolled opening of hemichannels in the plasma membrane may be deleterious for the myocardium and blocking hemichannels may confer cardioprotection by preventing ionic imbalance, cell swelling and loss of critical metabolites. Currently, all known hemichannel inhibitors also block GJ channels, thereby disturbing electrical cell-cell communication. Here we aimed to characterize a nonapeptide, called Gap19, derived from the cytoplasmic loop (CL) of Cx43 as a hemichannel blocker and examined its effect on hemichannel currents in cardiomyocytes and its influence in cardiac outcome after ischemia/reperfusion. We report that Gap 19 inhibits Cx43 hemichannels without blocking GJ channels or Cx40/pannexin-1 hemichannels. Hemichannel inhibition is due to the binding of Gap19 to the C-terminus (CT) thereby preventing intramolecular CT-CL interactions. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 prevented metabolic inhibition-enhanced hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and modestly decreased the infarct size after myocardial ischemia/reperfusion in mice in vivo. We conclude that preventing Cx43 hemichannel opening with Gap19 confers limited protective effects against myocardial ischemia/reperfusion injury.

Manipulating connexin communication channels: use of peptidomimetics and the translational outputs.

Gap junctions are key components underpinning multicellularity. They provide cell to cell channel pathways that enable direct intercellular communication and cellular coordination in tissues and organs. The channels are constructed of a family of connexin (Cx) membrane proteins. They oligomerize inside the cell, generating hemichannels (connexons) composed of six subunits arranged around a central channel. After transfer to the plasma membrane, arrays of Cx hemichannels (CxHcs) interact and couple with partners in neighboring attached cells to generate gap junctions. Cx channels have been studied using a range of technical approaches. Short peptides corresponding to sequences in the extra- and intracellular regions of Cxs were used first to generate epitope-specific antibodies that helped studies on the organization and functions of gap junctions. Subsequently, the peptides themselves, especially Gap26 and -27, mimetic peptides derived from each of the two extracellular loops of connexin43 (Cx43), a widely distributed Cx, have been extensively applied to block Cx channels and probe the biology of cell communication. The development of a further series of short peptides mimicking sequences in the intracellular loop, especially the extremity of the intracellular carboxyl tail of Cx43, followed. The primary inhibitory action of the peptidomimetics occurs at CxHcs located at unapposed regions of the cell's plasma membrane, followed by inhibition of cell coupling occurring across gap junctions. CxHcs respond to a range of environmental conditions by increasing their open probability. Peptidomimetics provide a way to block the actions of CxHcs with some selectivity. Furthermore, they are increasingly applied to address the pathological consequences of a range of environmental stresses that are thought to influence Cx channel operation. Cx peptidomimetics show promise as candidates in developing new therapeutic approaches for containing and reversing damage inflicted on CxHcs, especially in hypoxia and ischemia in the heart and in brain functions.

Connexin hemichannels and gap junction channels are differentially influenced by lipopolysaccharide and basic fibroblast growth factor.

Gap junction (GJ) channels are formed by two hemichannels (connexons), each contributed by the cells taking part in this direct cell-cell communication conduit. Hemichannels that do not interact with their counterparts on neighboring cells feature as a release pathway for small paracrine messengers such as nucleotides, glutamate, and prostaglandins. Connexins are phosphorylated by various kinases, and we compared the effect of various kinase-activating stimuli on GJ channels and hemichannels. Using peptides identical to a short connexin (Cx) amino acid sequence to specifically block hemichannels, we found that protein kinase C, Src, and lysophosphatidic acid (LPA) inhibited GJs and hemichannel-mediated ATP release in Cx43-expressing C6 glioma cells (C6-Cx43). Lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) inhibited GJs, but they stimulated ATP release via hemichannels in C6-Cx43. LPS and bFGF inhibited hemichannel-mediated ATP release in HeLa-Cx43 cells, but they stimulated it in HeLa-Cx43 with a truncated carboxy-terminal (CT) domain or in HeLa-Cx26, which has a very short CT. Hemichannel potentiation by LPS was inhibited by blockers of the arachidonic acid metabolism, and arachidonic acid had a potentiating effect like LPS and bFGF. We conclude that GJ channels and hemichannels display similar or oppositely directed responses to modulatory influences, depending on the balance between kinase activity and the activity of the arachidonic acid pathway. Distinctive hemichannel responses to pathological stimulation with LPS or bFGF may serve to optimize the cell response, directed at strictly controlling cellular ATP release, switching from direct GJ communication to indirect paracrine signaling, or maximizing cell-protective strategies.

CD4+ T lymphocyte subsets express connexin 43 and establish gap junction channel communication with macrophages in vitro.

Gap junction channels constructed of connexins (Cxs) are expressed by peripheral and secondary lymphoid organ-derived lymphocytes. These channels in the plasma membrane play key roles in a range of lymphocyte functions exemplified by the synthesis and secretion of Igs and cytokines and during transmigration across the endothelium. Most recently, their involvement in antigen cross-presentation has also been established. We report here for the first time the expression of mRNA and protein encoding Cx43 in mouse-derived CD4+ Th0, Th1, and Th2 lymphocyte subpopulations and demonstrate the establishment gap junction channel formation with primary macrophages in vitro. We show that this mode of direct communication is particularly favored in Th1-macrophage interactions and that LPS inhibits lymphocyte-macrophage cross-talk independently of the subset of lymphocyte involved. Our work suggests that gap junction-mediated communication can be modulated in the absence of specific antigenic stimulation. Therefore, a further mechanism featuring gap junction-mediated communication may be implicated in immune regulation.

Adenosine stimulates connexin 43 expression and gap junctional communication in pituitary folliculostellate cells.

Adenosine is known to stimulate interleukin (IL)-6 and vascular endothelial growth factor (VEGF) secretion from pituitary TtT/GF folliculostellate [corrected] (FS) cells indicating that it is an important paracrine regulator of anterior pituitary function. This study demonstrates that rodent anterior pituitary cell lines produce extracellular adenosine that is able to increase intercellular gap junction communication in FS cells. Ecto-5'-nucleotidase (CD73), the enzyme that generates adenosine from AMP, was demonstrated by immunocytochemistry in approximately 20% of anterior pituitary cells, and some of these cells colocalized with prolactin and growth hormone. CD73 mRNA and protein were detected in GH3 and MMQ (somatotroph-lactotroph lineages) and TtT/GF cells, and enzyme activity was demonstrated by the conversion of exogenously added fluorescent ethenoAMP to ethenoadenosine. Adenosine production, as measured by HPLC, was detected in GH3 (1 microM/h) and MMQ (3 microM/h) but not in TtT/GF cells. Adenosine (EC50: 0.5 microM) and NECA (universal adenosine receptor agonist; EC50 0.1 microM) stimulated connexin 43 (Cx43) mRNA and protein expression within 1-2 h in TtT/GF cells. Adenosine and NECA also stimulated gap junctional intercellular communication (as assessed by transmission of Alexa Fluor 488) by 6- to 8-fold in comparison with untreated TtT/GF cells. In cocultures of MMQ and TtT/GF cells, Cx43 expression in TtT/GF cells increased in proportion to the number of MMQ cells plated out. These data suggest that adenosine, formed locally in the anterior pituitary gland can stimulate gap junction communication in FS cells.

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.

The antiarrhythmic peptide rotigaptide (ZP123) increases gap junction intercellular communication in cardiac myocytes and HeLa cells expressing connexin 43.

We investigated the effects of rotigaptide (ZP123), a stable hexapeptide with antiarrhythmic properties, on gap junction mediated intercellular communication in contracting rat neonatal cardiac myocytes, HL-1 cells derived from cardiac atrium and in HeLa cells transfected with cDNA encoding Cx43-GFP, Cx32-GFP, Cx26-GFP, wild-type Cx43 or wild-type Cx26. Intercellular communication was monitored before and after treatment with rotigaptide following microinjection of small fluorescent dyes (MW<1 kDa). The communication-modifying effect of rotigaptide was confined to cells expressing Cx43 since the peptide had no effect on dye transfer in HeLa cells expressing Cx32-GFP, Cx26-GFP or wild-type Cx26. In contrast, HeLa cells expressing Cx43-GFP exposed to 50 nM rotigaptide for 5 h showed a 40% increase in gap junction mediated communication. Rotigaptide (50 nM) increased intercellular dye transfer in myocytes and atrial HL-1 cells, where Cx43 is the dominant connexin. However, it caused no change in cell beating rates of cardiac myocytes. Western blot analysis showed that rotigaptide did not modify the overall level of Cx43 expression and changes in the phosphorylation status of the protein were not observed.We conclude that the effects of rotigaptide were confined to cells expressing Cx43.

Reversible connexin 43 dephosphorylation during hypoxia and reoxygenation is linked to cellular ATP levels.

Altered gap junction coupling of cardiac myocytes during ischemia may contribute to development of lethal arrhythmias. The phosphoprotein connexin 43 (Cx43) is the major constituent of gap junctions. Dephosphorylation of Cx43 and uncoupling of gap junctions occur during ischemia, but the significance of Cx43 phosphorylation in this setting is unknown. Here we show that Cx43 dephosphorylation in synchronously contracting myocytes during ischemia is reversible, independent of hypoxia, and closely associated with cellular ATP levels. Cx43 became profoundly dephosphorylated during hypoxia only when glucose supplies were limited and was completely rephosphorylated within 30 minutes of reoxygenation. Similarly, direct reduction of ATP by various combinations of metabolic inhibitors and by ouabain was closely paralleled by loss of phosphoCx43 and recovery of phosphoCx43 accompanied restoration of ATP. Dephosphorylation of Cx43 could not be attributed to hypoxia, acid pH or secreted metabolites, or to AMP-activated protein kinase; moreover, the process was selective for Cx43 because levels of phospho-extracellular signal regulated kinase (ERK)1/2 were increased throughout. Rephosphorylation of Cx43 was not dependent on new protein synthesis, or on activation of protein kinases A or G, ERK1/2, p38 mitogen-activated protein kinase, or Jun kinase; however, broad-spectrum protein kinase C inhibitors prevented Cx43 rephosphorylation while also sensitizing myocytes to reoxygenation-mediated cell death. We conclude that Cx43 is reversibly dephosphorylated and rephosphorylated during hypoxia and reoxygenation by a novel mechanism that is sensitive to nonlethal fluctuations in cellular ATP. The role of this regulated phosphorylation in the adaptation to ischemia remains to be determined.

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.

Incorporation of connexins into plasma membranes and gap junctions.

Gap junctions are polymeric assemblies of aligned pairs of interacting hexameric connexon hemichannel units facilitating direct intercellular communication. The principal process leading to assembly of gap junctions involves the cotranslational insertion of connexin (Cx) proteins into the endoplasmic reticulum, followed by their rapid oligomeric association into homo- or heteromeric connexons that are trafficked via the Golgi apparatus to the plasma membrane. Oligomerisation is a high-fidelity process that determines connexon channel stoichiometry and conductance characteristics. A large number of mutations in Cx26 and Cx32 detected in genetic diseases have emphasised the requirement for precise oligomerisation of connexins into hexameric connexons that traffic to the plasma membrane. Mutations in Cx43 are rare, and in the cardiovascular system, where it is the dominant connexin, disease changes are linked to its abundance and to gap junction remodelling. Connexins with short carboxyl tails may also be post-translationally inserted as oligomeric channels directly into plasma membranes. This mechanism of channel assembly is highly dependent on microtubule integrity and may allow cells to rapidly modulate gap junctional cross talk.

Photoliberating inositol-1,4,5-trisphosphate triggers ATP release that is blocked by the connexin mimetic peptide gap 26.

Calcium signals can be communicated between cells by the diffusion of a second messenger through gap junction channels or by the release of an extracellular purinergic messenger. We investigated the contribution of these two pathways in endothelial cell lines by photoliberating InsP(3) or calcium from intracellular caged precursors, and recording either the resulting intercellular calcium wave or else the released ATP with a luciferin/luciferase assay. Photoliberating InsP(3) in a single cell within a confluent culture triggered an intercellular calcium wave, which was inhibited by the gap junction blocker alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptide gap 26, the purinergic inhibitors suramin, PPADS and apyrase and by purinergic receptor desensitisation. InsP(3)-triggered calcium waves were able to cross 20 microm wide cell-free zones. Photoliberating InsP(3) triggered ATP release that was blocked by buffering intracellular calcium with BAPTA and by applying gap 26. Gap 26, however, did not inhibit the gap junctional coupling between the cells as measured by fluorescence recovery after photobleaching. Photoliberating calcium did not trigger intercellular calcium waves or ATP release. We conclude that InsP(3)-triggered ATP release through connexin hemichannels contributes to the intercellular propagation of calcium signals.

Epidermal stem cells do not communicate through gap junctions.

Although enrichment of putative epidermal stem cells has been achieved, a need for additional markers that can enable isolation of live keratinocytes is crucial for characterization of these cells. Earlier work has shown that connexin proteins are absent from basal cells in the limbal epithelium, a region of the corneal epithelium enriched in corneal stem cells. Accordingly, we investigated whether connexin 43, a gap junction protein present in the basal layer of normal human epidermis, can serve as a negative marker for keratinocyte stem cells. In humans, cells with immunohistochemically undetectable levels of connexin 43 are found in the epidermal basal layer of neonatal foreskin and in the follicular bulge region. About 10% of the basal keratinocytes are connexin 43 negative, as determined by flow cytometry. These cells are uniformly small and low in granularity. Restricted gap junction communication was confirmed by the failure of low molecular weight dyes to transfer between cells. Experiments carried out in mouse epidermis demonstrated that most of the slowly cycling cells, detected as label-retaining cells, do not express connexin 43. Thus, presumptive keratinocyte stem cells can be identified and separated based on connexin 43 expression.

Connexin channels, connexin mimetic peptides and ATP release.

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP(3) elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.

Control of the proliferation of activated CD4+ T cells by connexins.

As expression of Cxs in cells of the immune system increases upon cellular activation, we investigated whether Cxs and especially CxHcs play a major role during T cell-mediated responses. In particular, we studied the expression of Cx43Hc following CD4(+) T cell stimulation using flow cytometry, real-time PCR, and Western blot analysis. We showed that expression of Cx43 and its phosphorylated isoforms increased in response to the engagement of CD3 and CD28. Cx43Hcs were found to be involved in sustaining proliferation of T cells, as assessed by cell cycle staining, thymidine incorporation assays, and CFSE analysis of cells exposed to mimetic peptide inhibitors of the plasma membrane Cx channels and antibodies generated to an extracellular region of Cx. The reduction of T cell proliferation mediated by Cx channel inhibitors suppressed cysteine uptake but not cytokine production. We conclude that upon antigen recognition, T cells require CxHc to sustain their clonal expansion.

Low connexin channel-dependent intercellular communication in human adult hematopoietic progenitor/stem cells: probing mechanisms of autologous stem cell therapy.

Human bone marrow is a clinical source of autologous progenitor stem cells showing promise for cardiac repair following ischemic insult. Functional improvements following delivery of adult bone marrow CD34(+) cells into heart tissue may require metabolic/electrical communication between participating cells. Since connexin43 (Cx43) channels are implicated in cardiogenesis and provide intercellular connectivity in the heart, the authors analyzed the expression of 20 connexins (Cx) in CD34(+) cells and in monocytes and granulocytes in bone marrow and spinal cord. Reverse transcriptase-polymerase chain reaction (RT-PCR) detected only low expression of Cx43 and Cx37. Very low level dye coupling was detected by flow cytometry between CD34(+) cells and other Cx43 expressing cells, including HL-1 cardiac cells, and was not inhibited by specific gap junction inhibitors. The results indicate that CD34(+) cells are unlikely to communicate via gap junctions and the authors conclude that use of CD34(+) cells to repair damaged hearts is unlikely to involve gap junctions. The results concur with the hypothesis that bone marrow cells elicit improved cardiac function through release of undefined paracrine mediators.

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.

ATP release by cardiac myocytes in a simulated ischaemia model: inhibition by a connexin mimetic and enhancement by an antiarrhythmic peptide.

We studied the role of connexin hemichannels in the release of ATP by neonatal cardiac myocytes subject to ischaemic stress. Mechanical, osmotic and oxidative stress and changes in extracellular or intracellular Ca(2+) levels induce connexin hemichannels located in the plasma membrane to open and release small ions and molecules with signaling potential such as ATP. Since ATP release has been implicated in adaptation to oxygen deprivation, we studied its release by cardiac myocytes incubated in a custom-built hypoxia chamber for various periods. In a simulated ischaemia model (0.5% oxygen and 0.2 g/l glucose) a peak of ATP release occurred at 80 min followed by a return to steady state levels for a further 200 min. This peak of ATP release was not observed in myocytes subject to hypoxia (0.5% oxygen, 3.0 g/l glucose). ATP release in ischaemia was influenced by two classes of reagents that target connexins, the channel forming proteins of gap junctions. First, the connexin hemichannel inhibitors Gap 26 and 18a glycyrrhetinic acid abolished the ATP peak of release. Second, the AAP10, a peptide with antiarrhythmic properties markedly increased the peak of ATP release observed at 80 min of ischaemia and also induced a second smaller peak at 180-240 min. ATP content of the myocytes and Cx43 phosphorylation were monitored. Since the release of ATP in ischaemia was abolished by connexin channel inhibitors and stimulated by a peptide developed to target connexins in the context of cardiac arrhythmia, the results suggest that nucleotide release by connexin hemichannels is likely to feature in the response of myocytes to ischaemic stress in the heart.

Ca(2+) regulation of connexin 43 hemichannels in C6 glioma and glial cells.

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca(2+) ([Ca(2+)](i)) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca(2+)](i) was in the 500nM range but the responses disappeared with larger [Ca(2+)](i) transients. Ca(2+)-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca(2+)-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca(2+)](i) triggers hemichannel opening, not by a direct action of Ca(2+) on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca(2+)](i) and acting to restrain cellular ATP loss.

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.