Cx43-hemichannel function and regulation in physiology and pathophysiology: insights from the bovine corneal endothelial cell system and beyond

Intercellular communication in primary bovine corneal endothelial cells (BCECs) is mainly driven by the release of extracellular ATP through Cx43 hemichannels. Studying the characteristics of Ca2+-wave propagation in BCECs, an important form of intercellular communication, in response to physiological signaling events has led to the discovery of important insights in the functional properties and regulation of native Cx43 hemichannels. Together with ectopic expression models for Cx43 hemichannels and truncated/mutated Cx43 versions, it became very clear that loop/tail interactions play a key role in controlling the activity of Cx43 hemichannels. Interestingly, the negative regulation of Cx43 hemichannels by enhanced actin/myosin contractility seems to impinge upon loss of these loop/tail interactions essential for opening Cx43 hemichannels. Finally, these molecular insights have spurred the development of novel peptide tools that can selectively inhibit Cx43 hemichannels, but neither Cx43 gap junctions nor hemichannels formed by other Cx isoforms. These tools now set the stage to hunt for novel physiological functions for Cx43 hemichannels in primary cells and tissues and to tackle disease conditions associated with excessive, pathological Cx43-hemichannel openings

Intercellular Ca2+ waves: mechanisms and functions

Intercellular calcium (Ca2+) waves (ICWs) represent the propagation of increases in intracellular Ca2+ through a syncytium of cells and appear to be a fundamental mechanism for coordinating multicellular responses. ICWs occur in a wide diversity of cells and have been extensively studied in vitro. More recent studies focus on ICWs in vivo. ICWs are triggered by a variety of stimuli and involve the release of Ca2+ from internal stores. The propagation of ICWs predominately involves cell communication with internal messengers moving via gap junctions or extracellular messengers mediating paracrine signaling. ICWs appear to be important in both normal physiology as well as pathophysiological processes in a variety of organs and tissues including brain, liver, retina, cochlea, and vascular tissue. We review here the mechanisms of initiation and propagation of ICWs, the key intra-and extracellular messengers (inositol 1,4,5-trisphosphate and ATP) mediating ICWs, and the proposed physiological functions of ICWs

Connexins : substrates and regulators of autophagy

Connexins mediate intercellular communication by assembling into hexameric channel complexes that act as hemichannels and gap junction channels. Most connexins are characterized by a very rapid turn-over in a variety of cell systems. The regulation of connexin turn-over by phosphorylation and ubiquitination events has been well documented. Moreover, different pathways have been implicated in connexin degradation, including proteasomal and lysosomal-based pathways. Only recently, autophagy emerged as an important connexin-degradation pathway for different connexin isoforms. As such, conditions well known to induce autophagy have an immediate impact on the connexin-expression levels. This is not only limited to experimental conditions but also several pathophysiological conditions associated with autophagy (dys) function affect connexin levels and their presence at the cell surface as gap junctions. Finally, connexins are not only substrates of autophagy but also emerge as regulators of the autophagy process. In particular, several connexin isoforms appear to recruit pre-autophagosomal autophagy-related proteins, including Atg16 and PI3K-complex components, to the plasma membrane, thereby limiting their availability and capacity for regulating autophagy

Inhibition of Connexin43 hemichannels impairs spatial short-term memory without affecting spatial working memory

Astrocytes are active players in higher brain function as they can release gliotransmitters, which are essential for synaptic plasticity. Various mechanisms have been proposed for gliotransmission, including vesicular mechanisms as well as non-vesicular ones, for example by passive diffusion via connexin hemichannels (HCs). We here investigated whether interfering with connexin43 (Cx43) HCs influenced hippocampal spatial memory. We made use of the peptide Gap19 that blocks HCs but not gap junction channels and is specific for Cx43. To this end, we microinfused transactivator of transcription linked Gap19 (TAT-Gap19) into the brain ventricle of male NMRI mice and assessed spatial memory in a Y maze. We found that the in vivo blockade of Cx43 HCs did not affect the locomotor activity or spatial working memory in a spontaneous alternation Y maze task. Cx43 blockade did however significantly impair the spatial short-term memory in a delayed spontaneous alternation Y maze task. These results indicate that Cx43 HCs play a role in spatial short-term memory

Astrocytic Connexin43 channels as candidate targets in epilepsy treatment

In epilepsy research, emphasis is put on exploring non-neuronal targets such as astrocytic proteins, since many patients remain pharmacoresistant to current treatments, which almost all target neuronal mechanisms. This paper reviews available data on astrocytic connexin43 (Cx43) signaling in seizures and epilepsy. Cx43 is a widely expressed transmembrane protein and the constituent of gap junctions (GJs) and hemichannels (HCs), allowing intercellular and extracellular communication, respectively. A plethora of research papers show altered Cx43 mRNA levels, protein expression, phosphorylation state, distribution and/or functional coupling in human epileptic tissue and experimental models. Human Cx43 mutations are linked to seizures as well, as 30% of patients with oculodentodigital dysplasia (ODDD), a rare genetic condition caused by mutations in the GJA1 gene coding for Cx43 protein, exhibit neurological symptoms including seizures. Cx30/Cx43 double knock-out mice show increased susceptibility to evoked epileptiform events in brain slices due to impaired GJ-mediated redistribution of K+ and glutamate and display a higher frequency of spontaneous generalized chronic seizures in an epilepsy model. Contradictory, Cx30/Cx43 GJs can traffic nutrients to high-energy demanding neurons and initiate astrocytic Ca2+ waves and hyper synchronization, thereby supporting proconvulsant effects. The general connexin channel blocker carbenoxolone and blockers from the fenamate family diminish epileptiform activity in vitro and improve seizure outcome in vivo. In addition, interventions with more selective peptide inhibitors of HCs display anticonvulsant actions. To conclude, further studies aiming to disentangle distinct roles of HCs and GJs are necessary and tools specifically targeting Cx43 HCs may facilitate the search for novel epilepsy treatments

Connexin-dependent neuroglial networking as a new therapeutic target

Astrocytes and neurons dynamically interact during physiological processes, and it is now widely accepted that they are both organized in plastic and tightly regulated networks. Astrocytes are connected through connexin-based gap junction channels, with brain region specificities, and those networks modulate neuronal activities, such as those involved in sleep-wake cycle, cognitive, or sensory functions. Additionally, astrocyte domains have been involved in neurogenesis and neuronal differentiation during development; they participate in the "tripartite synapse" with both pre-synaptic and post-synaptic neurons by tuning down or up neuronal activities through the control of neuronal synaptic strength. Connexin-based hemichannels are also involved in those regulations of neuronal activities, however, this feature will not be considered in the present review. Furthermore, neuronal processes, transmitting electrical signals to chemical synapses, stringently control astroglial connexin expression, and channel functions. Long-range energy trafficking toward neurons through connexin-coupled astrocytes and plasticity of those networks are hence largely dependent on neuronal activity. Such reciprocal interactions between neurons and astrocyte networks involve neurotransmitters, cytokines, endogenous lipids, and peptides released by neurons but also other brain cell types, including microglial and endothelial cells. Over the past 10 years, knowledge about neuroglial interactions has widened and now includes effects of CNS-targeting drugs such as antidepressants, antipsychotics, psychostimulants, or sedatives drugs as potential modulators of connexin function and thus astrocyte networking activity. In physiological situations, neuroglial networking is consequently resulting from a two-way interaction between astrocyte gap junction-mediated networks and those made by neurons. As both cell types are modulated by CNS drugs we postulate that neuroglial networking may emerge as new therapeutic targets in neurological and psychiatric disorders

Neurological manifestations of oculodentodigital dysplasia: a Cx43 channelopathy of the central nervous system?

The coordination of tissue function is mediated by gap junctions (GJs) that enable direct cell–cell transfer of metabolic and electric signals. GJs are formed by connexins of which Cx43 is most widespread in the human body. In the brain, Cx43 GJs are mostly found in astroglia where they coordinate the propagation of Ca2+ waves, spatial K+ buffering, and distribution of glucose. Beyond its role in direct intercellular communication, Cx43 also forms unapposed, non-junctional hemichannels in the plasma membrane of glial cells. These allow the passage of several neuro- and gliotransmitters that may, combined with downstream paracrine signaling, complement direct GJ communication among glial cells and sustain glial-neuronal signaling. Mutations in the GJA1 gene encoding Cx43 have been identified in a rare, mostly autosomal dominant syndrome called oculodentodigital dysplasia (ODDD). ODDD patients display a pleiotropic phenotype reflected by eye, hand, teeth, and foot abnormalities, as well as craniofacial and bone malformations. Remarkably, neurological symptoms such as dysarthria, neurogenic bladder (manifested as urinary incontinence), spasticity or muscle weakness, ataxia, and epilepsy are other prominent features observed in ODDD patients. Over 10 mutations detected in patients diagnosed with neurological disorders are associated with altered functionality of Cx43 GJs/hemichannels, but the link between ODDD-related abnormal channel activities and neurologic phenotype is still elusive. Here, we present an overview on the nature of the mutants conveying structural and functional changes of Cx43 channels and discuss available evidence for aberrant Cx43 GJ and hemichannel function. In a final step, we examine the possibilities of how channel dysfunction may lead to some of the neurological manifestations of ODDD

Connexin 43 in Mitochondria: What Do We Really Know About Its Function?

Connexin: Ischemia-reperfusion injury; MitochondriaConexina; Lesión por isquemia-reperfusión; MitocondriasConnexina; Lesió per isquèmia-reperfusió; MitocondrisConnexins are known for their ability to mediate cell-cell communication via gap junctions and also form hemichannels that pass ions and molecules over the plasma membrane when open. Connexins have also been detected within mitochondria, with mitochondrial connexin 43 (Cx43) being the best studied to date. In this review, we discuss evidence for Cx43 presence in mitochondria of cell lines, primary cells and organs and summarize data on its localization, import and phosphorylation status. We further highlight the influence of Cx43 on mitochondrial function in terms of respiration, opening of the mitochondrial permeability transition pore and formation of reactive oxygen species, and also address the presence of a truncated form of Cx43 termed Gja1-20k. Finally, the role of mitochondrial Cx43 in pathological conditions, particularly in the heart, is discussed.KB is funded by the German Research Foundation (BO 2955/4-1). LL is supported by the Research Foundation Flanders (FWO) grant numbers G.0527.18N and G040720N. MR-M is supported by the Instituto de Salud Carlos III of the Spanish Ministry of Health (FIS-PI19-01196) and a grant from the Sociedad Española de Cardiología (SEC/FEC-INV- BAS 217003)

RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho) physiological conditions of stress

Single and fractionated ionizing radiation induce alterations in endothelial connexin expression and channel function

Radiotherapy is an effective treatment for most tumor types. However, emerging evidence indicates an increased risk for atherosclerosis after ionizing radiation exposure, initiated by endothelial cell dysfunction. Interestingly, endothelial cells express connexin (Cx) proteins that are reported to exert proatherogenic as well as atheroprotective effects. Furthermore, Cxs form channels, gap junctions and hemichannels, that are involved in bystander signaling that leads to indirect radiation effects in non-exposed cells. We here aimed to investigate the consequences of endothelial cell irradiation on Cx expression and channel function. Telomerase immortalized human Coronary Artery/Microvascular Endothelial cells were exposed to single and fractionated X-rays. Several biological endpoints were investigated at different time points after exposure: Cx gene and protein expression, gap junctional dye coupling and hemichannel function. We demonstrate that single and fractionated irradiation induce upregulation of proatherogenic Cx43 and downregulation of atheroprotective Cx40 gene and protein levels in a dose-dependent manner. Single and fractionated irradiation furthermore increased gap junctional communication and induced hemichannel opening. Our findings indicate alterations in Cx expression that are typically observed in endothelial cells covering atherosclerotic plaques. The observed radiation-induced increase in Cx channel function may promote bystander signaling thereby exacerbating endothelial cell damage and atherogenesis