Hemichannel Opening Research Articles

Carboxyfluorescein Dye Uptake to Measure Connexin-mediated Hemichannel Activity in Cultured Cells

Connexins are membrane bound proteins that facilitate direct and local paracrine mediated cell-to-cell communication through their ability to oligomerise into hexameric hemichannels. When neighbouring channels align, they form gap-junctions that provide a direct route for information transfer between cells. In contrast to intact gap junctions, which typically open under physiological conditions, undocked hemichannels have a low open probability and mainly open in response to injury. Hemichannels permit the release of small molecules and ions (approximately 1kDa) into the local intercellular environment, and excessive expression/activity has been linked to a number of disease conditions. Carboxyfluorescein dye uptake measures functional expression of hemichannels, where increased hemichannel activity/function reflects increased loading. The technique relies on the uptake of a membrane-impermeable fluorescent tracer through open hemichannels, and can be used to compare channel activity between cell monolayers cultured under different conditions, e.g. control versus disease. Other techniques, such as biotinylation and electrophysiology can measure cell surface expression and hemichannel open probability respectively, however, carboxyfluorescein uptake provides a simple, rapid and cost-effective method to determine hemichannel activity in vitro in multiple cell types. Graphic abstract: Using dye uptake to measure hemichannel activity.

Tonabersat Inhibits Connexin43 Hemichannel Opening and Inflammasome Activation in an In Vitro Retinal Epithelial Cell Model of Diabetic Retinopathy.

This study was undertaken to evaluate the connexin hemichannel blocker tonabersat for the inhibition of inflammasome activation and use as a potential treatment for diabetic retinopathy. Human retinal pigment epithelial cells (ARPE-19) were stimulated with hyperglycemia and the inflammatory cytokines IL-1β and TNFα in order to mimic diabetic retinopathy molecular signs in vitro. Immunohistochemistry was used to evaluate the effect of tonabersat treatment on NLRP3, NLRP1, and cleaved caspase-1 expression and distribution. A Luminex cytokine release assay was performed to determine whether tonabersat affected proinflammatory cytokine release. NLRP1 was not activated in ARPE-19 cells, and IL-18 was not produced under disease conditions. However, NLRP3 and cleaved caspase-1 complex formation increased with hyperglycemia and cytokine challenge but was inhibited by tonabersat treatment. It also prevented the release of proinflammatory cytokines IL-1β, VEGF, and IL-6. Tonabersat therefore has the potential to reduce inflammasome-mediated inflammation in diabetic retinopathy.

Bracovirus-Mediated Innexin-Hemichannel Closure in Cell Disassembly

​Cell-cell communication is required for the cellular immune response. Immunosuppression occurs when polydnavirus virions are injected by parasitic wasps into caterpillar hosts. However, the effects of hemichannel closure on cellular immune response are not fully understood. Here, we examined apoptotic body formation triggered by hemichannel closure in haemocytes of the host, Spodoptera litura, infected with bracovirus from the parasitic wasp, Microplitis bicoloratus. We show that Microplitis bicoloratus bracovirus (MbBV) induced apoptotic cell disassembly and apoptotic body formation, accompanied by hemichannel closure. Haemocyte apoptotic body formation was caused by dysregulation of innexins (Inxs) 1‒4 during MbBV-mediated inhibition of pI3K/AKT signalling and activation of protein tyrosine phosphatase 109. Mechanistically, MbBV activated caspase 3, which cleaved gap junction Inx proteins. Our results show that hemichannel opening and closure by various stimuli causing modulation of the levels of the gap junction proteins, Inxs, could inhibit or activate apoptotic body formation, respectively.

Mechanosensitive collaboration between integrins and connexins allows nutrient and antioxidant transport into the lens.

The delivery of glucose and antioxidants is vital to maintain homeostasis and lens transparency. Here, we report a new mechanism whereby mechanically activated connexin (Cx) hemichannels serve as a transport portal for delivering glucose and glutathione (GSH). Integrin α6β1 in outer cortical lens fiber activated by fluid flow shear stress (FFSS) induced opening of hemichannels. Inhibition of α6 activation prevented hemichannel opening as well as glucose and GSH uptake. The activation of integrin β1, a heterodimeric partner of α6 in the absence of FFSS, increased Cx50 hemichannel opening. Hemichannel activation by FFSS depended on the interaction of integrin α6 and Cx50 C-terminal domain. Moreover, hemichannels in nuclear fiber were unresponsive owing to Cx50 truncation. Taken together, these results show that mechanically activated α6β1 integrin in outer cortical lens fibers leads to opening of hemichannels, which transport glucose and GSH into cortical lens fibers. This study unveils a new transport mechanism that maintains metabolic and antioxidative function of the lens.

Targeting connexin 43 provides anti-inflammatory effects after intracerebral hemorrhage injury by regulating YAP signaling

BackgroundIn the central nervous system (CNS), connexin 43 (Cx43) is mainly expressed in astrocytes and regulates astrocytic network homeostasis. Similar to Cx43 overexpression, abnormal excessive opening of Cx43 hemichannels (Cx43Hcs) on reactive astrocytes aggravates the inflammatory response and cell death in CNS pathologies. However, the role of excessive Cx43Hc opening in intracerebral hemorrhage (ICH) injury is not clear.MethodsHemin stimulation in primary cells and collagenase IV injection in C57BL/6J (B6) mice were used as ICH models in vitro and in vivo. After ICH injury, the Cx43 mimetic peptide Gap19 was used for treatment. Ethidium bromide (EtBr) uptake assays were used to measure the opening of Cx43Hcs. Western blotting and immunofluorescence were used to measure protein expression. qRT-PCR and ELISA were used to determine the levels of cytokines. Coimmunoprecipitation (Co-IP) and the Duolink in situ proximity ligation assay (PLA) were applied to measure the association between proteins.ResultsIn this study, Cx43 expression upregulation and excessive Cx43Hc opening was observed in mice after ICH injury. Delayed treatment with Gap19 significantly alleviated hematoma volume and neurological deficits after ICH injury. In addition, Gap19 decreased inflammatory cytokine levels in the tissue surrounding the hematoma and decreased reactive astrogliosis after ICH injury in vitro and in vivo. Intriguingly, Cx43 transcriptional activity and expression in astrocytes were significantly increased after hemin stimulation in culture. However, Gap19 treatment downregulated astrocytic Cx43 expression through the ubiquitin-proteasome pathway without affecting Cx43 transcription. Additionally, our data showed that Gap19 increased Yes-associated protein (YAP) nuclear translocation. This subsequently upregulated SOCS1 and SOCS3 expression and then inhibited the TLR4-NFκB and JAK2-STAT3 pathways in hemin-stimulated astrocytes. Finally, the YAP inhibitor, verteporfin (VP), reversed the anti-inflammatory effect of Gap19 in vitro and almost completely blocked its protective effects in vivo after ICH injury.ConclusionsThis study provides new insight into potential treatment strategies for ICH injury involving astroglial Cx43 and Cx43Hcs. Suppression of abnormal astroglial Cx43 expression and Cx43Hc opening by Gap19 has anti-inflammatory and neuroprotective effects after ICH injury.

Opposing modulation of Cx26 gap junctions and hemichannels by CO2.

A moderate increase in (55mmHg) closes Cx26 gap junctions. This effect of CO2 is independent of changes in intra- or extracellular pH. The CO2 -dependent closing effect depends on the same residues (K125 and R104) that are required for the CO2 -dependent opening of Cx26 hemichannels. Pathological mutations of Cx26 abolish the CO2 -dependent closing of the gap junction. Elastic network modelling suggests that the effect of CO2 on Cx26 hemichannels and gap junctions is mediated through changes in the lowest entropy state of the protein. Cx26 hemichannels open in response to moderate elevations of CO2 ( 55mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated of 55mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2 . Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 66.7%, 95% CI, 50.5-100.0%) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification. Using 30mm propionate, we found that acidification in the absence of a change in caused a median reduction in the gap junction conductance of 41.7% (95% CI, 26.6-53.7%). This effect of propionate was unaffected by the K125R mutation (median reduction 48.1%, 95% CI, 28.0-86.3%). pH-dependent and CO2 -dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the keratitis ichthyosis deafness syndrome (N14K, A40V and A88V), in combination with the mutation M151L, also abolished the CO2 -dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction.

Gap19, a Cx43 Hemichannel Inhibitor, Acts as a Gating Modifier That Decreases Main State Opening While Increasing Substate Gating.

Cx43 hemichannels (HCs) are electrically and chemically gated transmembrane pores with low open probability and multiple conductance states, which makes kinetic studies of channel gating in large datasets challenging. Here, we developed open access software, named HemiGUI, to analyze HC gating transitions and investigated voltage-induced HC opening based on up to ≈4000 events recorded in HeLa-Cx43-overexpressing cells. We performed a detailed characterization of Cx43 HC gating profiles and specifically focused on the role of the C-terminal tail (CT) domain by recording the impact of adding an EGFP tag to the Cx43 CT end (Cx43-EGFP) or by supplying the Cx43 HC-inhibiting peptide Gap19 that interferes with CT interaction with the cytoplasmic loop (CL). We found that Gap19 not only decreased HC opening activity to the open state (≈217 pS) but also increased the propensity of subconductance (≈80 pS) transitions that additionally became slower as compared to the control. The work demonstrates that large sample transition analysis allows detailed investigations on Cx43 HC gating and shows that Gap19 acts as a HC gating modifier by interacting with the CT that forms a crucial gating element.

Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function.

In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFPQL, by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.

Connexin Hemichannel Mimetic Peptide Attenuates Cortical Interneuron Loss and Perineuronal Net Disruption Following Cerebral Ischemia in Near-Term Fetal Sheep.

Perinatal hypoxia-ischemia is associated with disruption of cortical gamma-aminobutyric acid (GABA)ergic interneurons and their surrounding perineuronal nets, which may contribute to persisting neurological deficits. Blockade of connexin43 hemichannels using a mimetic peptide can alleviate seizures and injury after hypoxia-ischemia. In this study, we tested the hypothesis that connexin43 hemichannel blockade improves the integrity of cortical interneurons and perineuronal nets. Term-equivalent fetal sheep received 30 min of bilateral carotid artery occlusion, recovery for 90 min, followed by a 25-h intracerebroventricular infusion of vehicle or a mimetic peptide that blocks connexin hemichannels or by a sham ischemia + vehicle infusion. Brain tissues were stained for interneuronal markers or perineuronal nets. Cerebral ischemia was associated with loss of cortical interneurons and perineuronal nets. The mimetic peptide infusion reduced loss of glutamic acid decarboxylase-, calretinin-, and parvalbumin-expressing interneurons and perineuronal nets. The interneuron and perineuronal net densities were negatively correlated with total seizure burden after ischemia. These data suggest that the opening of connexin43 hemichannels after perinatal hypoxia-ischemia causes loss of cortical interneurons and perineuronal nets and that this exacerbates seizures. Connexin43 hemichannel blockade may be an effective strategy to attenuate seizures and may improve long-term neurological outcomes after perinatal hypoxia-ischemia.

Channels to consciousness: a possible role of gap junctions in consciousness.

The neurophysiological basis of consciousness is still unknown and one of the most challenging questions in the field of neuroscience and related disciplines. We propose that consciousness is characterized by the maintenance of mental representations of internal and external stimuli for the execution of cognitive operations. Consciousness cannot exist without working memory, and it is likely that consciousness and working memory share the same neural substrates. Here, we present a novel psychological and neurophysiological framework that explains the role of consciousness for cognition, adaptive behavior, and everyday life. A hypothetical architecture of consciousness is presented that is organized as a system of operation and storage units named platforms that are controlled by a consciousness center (central executive/online platform). Platforms maintain mental representations or contents, are entrusted with different executive functions, and operate at different levels of consciousness. The model includes conscious-mode central executive/online and mental time travel platforms and semiconscious steady-state and preconscious standby platforms. Mental representations or contents are represented by neural circuits and their support cells (astrocytes, oligodendrocytes, etc.) and become conscious when neural circuits reverberate, that is, fire sequentially and continuously with relative synchronicity. Reverberatory activity in neural circuits may be initiated and maintained by pacemaker cells/neural circuit pulsars, enhanced electronic coupling via gap junctions, and unapposed hemichannel opening. The central executive/online platform controls which mental representations or contents should become conscious by recruiting pacemaker cells/neural network pulsars, the opening of hemichannels, and promoting enhanced neural circuit coupling via gap junctions.

Connexins in neuromyelitis optica: a link between astrocytopathy and demyelination.

Neuromyelitis optica, a rare neuroinflammatory demyelinating disease of the CNS, is characterized by the presence of specific pathogenic autoantibodies directed against the astrocytic water channel aquaporin 4 (AQP4) and is now considered as an astrocytopathy associated either with complement-dependent astrocyte death or with astrocyte dysfunction. However, the link between astrocyte dysfunction and demyelination remains unclear. We propose glial intercellular communication, supported by connexin hemichannels and gap junctions, to be involved in demyelination process in neuromyelitis optica. Using mature myelinated cultures, we demonstrate that a treatment of 1 h to 48 h with immunoglobulins purified from patients with neuromyelitis optica (NMO-IgG) is responsible for a complement independent demyelination, compared to healthy donors' immunoglobulins (P < 0.001). In parallel, patients' immunoglobulins induce an alteration of connexin expression characterized by a rapid loss of astrocytic connexins at the membrane followed by an increased size of gap junction plaques (+60%; P < 0.01). This was co-observed with connexin dysfunction with gap junction disruption (-57%; P < 0.001) and increased hemichannel opening (+17%; P < 0.001), associated with glutamate release. Blocking connexin 43 hemichannels with a specific peptide was able to prevent demyelination in co-treatment with patients compared to healthy donors' immunoglobulins. By contrast, the blockade of connexin 43 gap junctions with another peptide was detrimental for myelin (myelin density -48%; P < 0.001). Overall, our results suggest that dysregulation of connexins would play a pathogenetic role in neuromyelitis optica. The further identification of mechanisms leading to connexin dysfunction and soluble factors implicated, would provide interesting therapeutic strategies for demyelinating disorders.

The Multifaceted Role of Astrocyte Connexin 43 in Ischemic Stroke Through Forming Hemichannels and Gap Junctions.

Ischemic stroke is a multi-factorial cerebrovascular disease with high worldwide morbidity and mortality. In the past few years, multiple studies have revealed the underlying mechanism of ischemia/reperfusion injury, including calcium overload, amino acid toxicity, oxidative stress, and inflammation. Connexin 43 (Cx43), the predominant connexin protein in astrocytes, has been recently proven to display non-substitutable roles in the pathology of ischemic stroke development and progression through forming gap junctions and hemichannels. Under normal conditions, astrocytic Cx43 could be found in hemichannels or in the coupling with other hemichannels on astrocytes, neurons, or oligodendrocytes to form the neuro–glial syncytium, which is involved in metabolites exchange between communicated cells, thus maintaining the homeostasis of the CNS environment. In ischemic stroke, the phosphorylation of Cx43 might cause the degradation of gap junctions and the opening of hemichannels, contributing to the release of inflammatory mediators. However, the remaining gap junctions could facilitate the exchange of protective and harmful metabolites between healthy and injured cells, protecting the injured cells to some extent or damaging the healthy cells depending on the balance of the exchange of protective and harmful metabolites. In this study, we review the changes in astrocytic Cx43 expression and distribution as well as the influence of these changes on the function of astrocytes and other cells in the CNS, providing new insight into the pathology of ischemic stroke injury; we also discuss the potential of astrocytic Cx43 as a target for the treatment of ischemic stroke.

Apocynin Treatment Prevents Cardiac Connexin 43 Hemichannels Hyperactivity by Reducing Nitroso-Redox Stress in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a fatal disease that causes cardiomyopathy and is associated with oxidative stress. In the heart, oxidative stress interferes with the location of connexin 43 (Cx43) to the intercalated discs causing its lateralization to the plasma membrane where Cx43 forms hemichannels. We tested the hypothesis that in DMD cardiomyopathy, increased oxidative stress is associated with the formation and activation of Cx43 hemichannels. For this, we used mdx mice as a DMD model and evaluated cardiac function, nitroso-redox changes and Cx43 hemichannels permeability. Mdx hearts presented increased NADPH oxidase-derived oxidative stress and increased Cx43 S-nitrosylation compared to controls. These redox changes were associated with increased Cx43 lateralization, decreased cardiac contractility and increased arrhythmic events. Pharmacological inhibition of NADPH oxidase using apocynin (one month) reduced systemic oxidative stress and reversed the aforementioned changes towards normal, except Cx43 lateralization. Opening of Cx43 hemichannels was blocked by apocynin treatment and by acute hemichannel blockade with carbenoxolone. NADPH oxidase inhibition also prevented the occurrence of apoptosis in mdx hearts and reversed the ventricular remodeling. These results show that NADPH oxidase activity in DMD is associated with S-nitrosylation and opening of Cx43 hemichannels. These changes lead to apoptosis and cardiac dysfunction and were prevented by NADPH oxidase inhibition.

Signaling Between TRPV1/TRPV4 and Intracellular Hydrostatic Pressure in the Mouse Lens.

PurposeThe lens uses feedback to maintain zero pressure in its surface cells. Positive pressures are detected by transient receptor potential vanilloid (TRPV4), which initiates a cascade that reduces surface cell osmolarity. The first step is opening of gap junction hemichannels. One purpose of the current study was to identify the connexin(s) in the hemichannels. Negative pressures are detected by TRPV1, which initiates a cascade that increases surface osmolarity. The increase in osmolarity was initially reported to be through inhibition of Na/K ATPase activity, but a recent study reported it was through stimulation of Na/K/2Cl (NKCC) cotransport. A second purpose of this study was to reconcile these two reports.MethodsIntracellular hydrostatic pressures were measured using a microelectrode/manometer system. Lenses from TRPV1 or Cx50 null mice were studied. Specific inhibitors of Cx50 gap junction channels, NKCC, and Akt were used.ResultsEither knockout of Cx50 or blockade of Cx50 channels completely eliminated the response to positive surface pressures. Knockout of Cx50 also caused a positive drift in surface pressure. The short-term (∼20-minute) response to negative surface pressures was eliminated by blockade of NKCC, but a long-term (∼4-hour) response restored pressure to zero. Both short- and long-term responses were eliminated by knockout of TRPV1 or inhibition of Akt.ConclusionsHemichannels made from Cx50 are required for the response to positive surface pressures. Negative surface pressures first activate NKCC, but a backup system is inhibition of Na/K ATPase activity. Both responses are initiated by TRPV1 and go through PI3K/Akt before branching.

Connexin Hemichannels Contribute to the Activation of cAMP Signaling Pathway and Renin Production.

Connexin hemichannels play an important role in the control of cellular signaling and behaviors. Given that lowering extracellular Ca2+, a condition that activates hemichannels, is a well-characterized stimulator of renin in juxtaglomerular cells, we, therefore, tested a potential implication of hemichannels in the regulation of renin in As4.1 renin-secreting cells. Lowering extracellular Ca2+ induced hemichannel opening, which was associated with cAMP signaling pathway activation and increased renin production. Blockade of hemichannels with inhibitors or downregulation of Cxs with siRNAs abrogated the activation of cAMP pathway and the elevation of renin. Further analysis revealed that cAMP pathway activation was blocked by adenylyl cyclase inhibitor SQ 22536, suggesting an implication of adenyl cyclase. Furthermore, the participation of hemichannels in the activation of the cAMP signaling pathway was also observed in a renal tubular epithelial cell line NRK. Collectively, our results characterized the hemichannel opening as a presently unrecognized molecular event involved in low Ca2+-elicited activation of cAMP pathway and renin production. Our findings thus provide novel mechanistic insights into the low Ca2+-initiated cell responses. Given the importance of cAMP signaling pathway in the control of multiple cellular functions, our findings also highlight the importance of Cx-forming channels in various pathophysiological situations.

Multi-walled carbon nanotubes induce IL-1β secretion by activating hemichannels-mediated ATP release in THP-1 macrophages

Multi-walled carbon nanotubes (MWCNTs) are known to induce pulmonary inflammatory effects through stimulating pro-inflammatory cytokine secretion from alveolar macrophages. Despite extensive studies on MWCNTs’ pro-inflammatory reactivity, the understanding of molecular mechanisms involved is still incomplete. In this study, we investigated hemichannel’s involvement in MWCNTs-induced macrophage IL-1β release. Our results showed that the unmodified and COOH MWCNTs could induce ATP release and ATP-P2X7R axis-dependent IL-1β secretion from THP-1 macrophages. By using various inhibitors, we confirmed that the MWCNTs-induced ATP release was primarily through hemichannels. EtBr dye uptake assay detected significant hemichannels opening in MWCNTs exposed THP-1 macrophages. Inhibition of hemichannels by CBX, 43Gap27, or 10Panx1 pretreatment results in decreased ATP and IL-1β release. The addition of ATP restored the reduced IL-1β secretion level from hemichannel inhibition. We also confirmed with five other types of MWCNTs that the induction of hemichannels by MWCNTs strongly correlates with their capacity to induce IL-1β secretion. Taken together, we conclude that hemichannels-mediated ATP release and subsequent NLRP3 inflammasome activation through P2X7R may be one mechanism by which MWCNTs induce macrophage IL-1β secretion. Our findings may provide a novel molecular mechanism for MWCNTs induced IL-1β secretion.

Xentry-Gap19 inhibits Connexin43 hemichannel opening especially during hypoxic injury.

Hypoxic injury results in cell death, tissue damage and activation of inflammatory pathways. This is mediated by pathological Connexin43 (Cx43) hemichannel (HC) opening resulting in osmotic and ionic imbalances as well as cytokine production perpetuating the inflammatory environment. Gap19 is an intracellularly acting Cx43 mimetic peptide that blocks HC opening and thus promotes cell survival. However, native Gap19, which must enter the cell in order to function, exhibits low cell permeability. In this study, Gap19 was conjugated to the cell-penetrating peptide, Xentry, to investigate if cellular uptake could be improved while maintaining peptide function. Cellular uptake of Xentry-Gap19 (XG19) was much greater than that of native Gap19 even under normal cell culture conditions. Peptide function was maintained post uptake as shown by reduced ethidium homodimer influx and ATP release due to Cx43 HC block. While XG19 blocked pathologic HC opening though, normal gap junction communication required for cell repair and survival mechanisms was not affected as shown in a dye scrape-load assay. Under hypoxic conditions, increased expression of Syndecan-4, a plasma membrane proteoglycan targeted by Xentry, enabled even greater XG19 uptake leading to higher inhibition of ATP release and greater cell survival. This suggests that XG19, which is targeted specifically to hypoxic cells, can efficiently and safely block Cx43 HC and could therefore be a novel treatment for hypoxic and inflammatory diseases. Graphical abstract

Interferon-γ and high glucose-induced opening of Cx43 hemichannels causes endothelial cell dysfunction and damage

Both IFN-γ or high glucose have been linked to systemic inflammatory imbalance with serious repercussions not only for endothelial function but also for the formation of the atherosclerotic plaque. Although the uncontrolled opening of connexin hemichannels underpins the progression of various diseases, whether they are implicated in endothelial cell dysfunction and damage evoked by IFN-γ plus high glucose remains to be fully elucidated. In this study, by using live cell imaging and biochemical approaches, we demonstrate that IFN-γ plus high glucose augment endothelial connexin43 hemichannel activity, resulting in the increase of ATP release, ATP-mediated Ca2+ dynamics and production of nitric oxide and superoxide anion, as well as impaired insulin-mediated uptake and intercellular diffusion of glucose and cell survival. Based on our results, we propose that connexin 43 hemichannel inhibition could serve as a new approach for tackling the activation of detrimental signaling resulting in endothelial cell dysfunction and death caused by inflammatory mediators during atherosclerosis secondary to diabetes mellitus.

HIV gp120 Protein Increases the Function of Connexin 43 Hemichannels and Pannexin-1 Channels in Astrocytes: Repercussions on Astroglial Function.

At least half of human immunodeficiency virus (HIV)-infected individuals suffer from a wide range of cognitive, behavioral and motor deficits, collectively known as HIV-associated neurocognitive disorders (HAND). The molecular mechanisms that amplify damage within the brain of HIV-infected individuals are unknown. Recently, we described that HIV augments the opening of connexin-43 (Cx43) hemichannels in cultured human astrocytes, which result in the collapse of neuronal processes. Whether HIV soluble viral proteins such as gp120, can regulate hemichannel opening in astrocytes is still ignored. These channels communicate the cytosol with the extracellular space during pathological conditions. We found that gp120 enhances the function of both Cx43 hemichannels and pannexin-1 channels in mouse cortical astrocytes. These effects depended on the activation of IL-1β/TNF-α, p38 MAP kinase, iNOS, cytoplasmic Ca2+ and purinergic signaling. The gp120-induced channel opening resulted in alterations in Ca2+ dynamics, nitric oxide production and ATP release. Although the channel opening evoked by gp120 in astrocytes was reproduced in ex vivo brain preparations, these responses were heterogeneous depending on the CA1 region analyzed. We speculate that soluble gp120-induced activation of astroglial Cx43 hemichannels and pannexin-1 channels could be crucial for the pathogenesis of HAND.

Novel animal model of multiple sclerosis: The glial connexin gap junction as an environmental tuner for neuroinflammation

AbstractConnexins, which comprise gap junctions (GJs) via homotypic/heterotypic oligomerization, act as channels to connect opposing cells, mainly in solid organs such as the skin, liver, heart, and central/peripheral nervous system. Connexins are synthesized in the endoplasmic reticulum, assembled in the Golgi apparatus as hexamers, and inserted into the cell membrane as hemichannels. These hemichannels are closed under normal conditions until they combine to form clusters and connect to neighboring cells via GJs in a head‐to‐head configuration. Opening of hemichannels, which depends on the intra‐ or extracellular environment, allows various bioactive molecules to enter into or be released from the host cells. Recent pathological studies on human demyelinating diseases have revealed alterations of connexin expression patterns in demyelinating lesions. To elucidate the molecular mechanisms of connexins in the pathomechanisms of inflammatory demyelination, we induced experimental autoimmune encephalomyelitis (EAE) in connexin 30 (Cx30)‐deficient mice, oligodendroglia‐specific Cx47‐ablated mice, and astroglia‐specific Cx43‐ablated mice. We found that both astroglial‐Cx30‐deficient mice and Cx43‐ablated mice showed amelioration of the clinical course of EAE, while oligodendroglial‐Cx47‐ablated mice showed aggravation. These findings indicate the distinct role of connexins expressed in different cell types and the substantial contribution of connexin‐mediated pathology to demyelinating disorders. The imbalance in connexin expression, which is caused by the inflammatory environment, results in an increase in hemichannels in glial cells. The release of proinflammatory molecules induced by the increase in hemichannels on activated glial cells is a crucial mechanism of demyelinating disorders.