Decoding Connexin Hemichannels: Structure, Function, and Regulatory Mechanisms.

Editorial: Single membrane channels formed by connexins or pannexins: focus on the nervous system

how atp and other nucleotides are released from intact cells is a fundamental question, given the existence of multiple purinergic receptor signaling cascades operative in most vertebrate tissues ([25][1]). It is well-established that neurons and neuroendocrine cells release ATP via classical

Connexin hemichannels: novel mediators of toxicity.

Simple SummaryConnexin hemichannels are formed by connexin protein family members, and connect cytosol and extracellular milium. The function of connexin hemichannels cannot be readily distinguished from the gap junctions formed also by connexins, or hemichannels formed by pannexins. It appears that connexin hemichannels are normally closed to maintain cellular homeostasis, but can be activated in pathophysiological processes to serve as toxic membrane pores. On the other hand, gap junctions are normally open in order to perform critical physiological functions, but are often closed or down-regulated under pathological conditions. The development and characterization of connexin mimetic peptides have resulted in a panel of connexin hemichannel-selective blockers. Investigations using these blockers have shown that the opening of connexin hemichannels facilitates the release of damage-associated molecular patterns, a class of endogenous molecules that are critical for the pathogenesis of inflammatory diseases. The blockade of connexin hemichannels virtually always leads to attenuated inflammation, reduced tissue injury and improved organ function. In this review, we provide an updated view of the role of connexin hemichannels in inflammatory diseases.The connexin protein family consists of approximately 20 members, and is well recognized as the structural unit of the gap junction channels that perforate the plasma membranes of coupled cells and, thereby, mediate intercellular communication. Gap junctions are assembled by two preexisting hemichannels on the membranes of apposing cells. Non-junctional connexin hemichannels (CxHC) provide a conduit between the cell interior and the extracellular milieu, and are believed to be in a protectively closed state under physiological conditions. The development and characterization of the peptide mimetics of the amino acid sequences of connexins have resulted in the development of a panel of blockers with a higher selectivity for CxHC, which have become important tools for defining the role of CxHC in various biological processes. It is increasingly clear that CxHC can be induced to open by pathogen-associated molecular patterns. The opening of CxHC facilitates the release of damage-associated molecular patterns, a class of endogenous molecules that are critical for the pathogenesis of inflammatory diseases. The blockade of CxHC leads to attenuated inflammation, reduced tissue injury and improved organ function in human and animal models of about thirty inflammatory diseases and disorders. These findings demonstrate that CxHC may contribute to the intensification of inflammation, and serve as a common target in the treatments of various inflammatory diseases. In this review, we provide an update on the progress in the understanding of CxHC, with a focus on the role of these channels in inflammatory diseases.

Progressive loss of neuronal structure and function occur in several neurodegenerative diseases. Cellular responses to brain injury depend on properties of the cells (e.g., hormonal nutritional status) and insult (e.g., duration, intensity, and quality), whereas, tissue responses depend on interactions between their constituent cells, including chemical and electrical transmission as well as paracrine and autocrine signaling. In vertebrate cells, autocrine and paracrine communication occur in part via release of chemical signals through connexin hemichannels (Saez et al. 2010), the precursors of gap junction channels that are formed by two hemichannels provided by one of each apposed cells (Fig. 1). Each hemichannel is composed of six protein subunits termed connexins, which are highly conserved proteins encoded by 21 genes in human and 20 in mouse with orthologs in other vertebrate species (Cruciani and Mikalsen 2005). Connexins are abundantly expressed in cells of the central nervous system (CNS) (Orellana et al. 2009) (Fig. 2), and they are named after their predicted molecular mass expressed in kDa, so that connexin43 (Cx43) has a molecular mass of ~43 kDa. For a long time the main function attributed to connexin hemichannels was the formation of gap junction channels. Nevertheless, in the last decade, the presence of functional connexin hemichannels in nonjunctional membranes has been demonstrated by several experimental approaches (Saez et al. 2010). These channels allow cellular release of relevant quantities of autocrine/paracrine signaling molecules (e.g., ATP, glutamate, NAD+ and PGE2) to the extracellular milieu (Bruzzone et al. 2001; Cherian et al. 2005; Stout et al. 2002), as well as uptake of small molecules (e.g., glucose) (Retamal et al. 2007a). Recently, another gene family encoding a set of three membrane proteins, named pannexins (Panxs 1-3), has been identified (Bruzzone et al. 2003). Up to now, only Panx3 has been shown to form gap junctions in osteoblasts (Ishikawa et al. 2011) and further studies will be required to identify pannexin gap junctions in other cell types. Connexins and pannexins present similar membrane topology, with four α-helical transmembrane domains connected by two

Connexin hemichannel inhibition reduces acetaminophen-induced liver injury in mice

Upon intravenous injection of tumour necrosis factor (TNF) in mice, a systemic inflammatory response syndrome (SIRS) is initiated, characterized by an acute cytokine storm and induction of vascular hyperpermeability. Connexin43 hemichannels have been implicated in various pathological conditions, e.g. ischemia and inflammation, and can lead to detrimental cellular outcomes. Here, we explored whether targeting connexin43 hemichannels could alleviate TNF-induced endothelial barrier dysfunction and lethality in SIRS. Therefore, we verified whether administration of connexin43-targeting-peptides affected survival, body temperature and vascular permeability in vivo. In vitro, TNF-effects on connexin43 hemichannel function were investigated by single-channel studies and Ca2+-imaging. Blocking connexin43 hemichannels with TAT-Gap19 protected mice against TNF-induced mortality, hypothermia and vascular leakage, while enhancing connexin43 hemichannel function with TAT-CT9 provoked opposite sensitizing effects. In vitro patch-clamp studies revealed that TNF acutely activated connexin43 hemichannel opening in endothelial cells, which was promoted by CT9, and inhibited by Gap19 and intracellular Ca2+-buffering. In vivo experiments aimed at buffering intracellular Ca2+, and pharmacologically targeting Ca2+/calmodulin-dependent protein kinase-II, a known modulator of endothelial barrier integrity, demonstrated their involvement in permeability alterations. Our results demonstrate significant benefits of inhibiting connexin43 hemichannels to counteract TNF-induced SIRS-associated vascular permeability and lethality.

Perinatal brain injury remains a major cause of death and life-long disability. Perinatal brain injury is typically associated with hypoxia-ischemia and/or infection/inflammation. Both hypoxia-ischemia and infection trigger an inflammatory response in the brain. The inflammatory response can contribute to brain cell loss and chronic neuroinflammation leading to neurological impairments. It is now well-established that brain injury evolves over time, and shows a striking spread from injured to previously uninjured regions of the brain. There is increasing evidence that this spread is related to opening of connexin hemichannels and pannexin channels, both of which are large conductance membrane channels found in almost all cell types in the brain. Blocking connexin hemichannels within the first 3 h after hypoxia-ischemia has been shown to improve outcomes in term equivalent fetal sheep but it is important to also understand the downstream pathways linking membrane channel opening with the development of injury in order to identify new therapeutic targets. Open membrane channels release adenosine triphosphate (ATP), and other neuroactive molecules, into the extracellular space. ATP has an important physiological role, but has also been reported to act as a damage-associated molecular pattern (DAMP) signal mediated through specific purinergic receptors and so act as a primary signal 1 in the innate immune system inflammasome pathway. More crucially, extracellular ATP is a key inflammasome signal 2 activator, with purinergic receptor binding triggering the assembly of the multi-protein inflammasome complex. The inflammasome pathway and complex formation contribute to activation of inflammatory caspases, and the release of inflammatory cytokines, including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-18, and vascular endothelial growth factor (VEGF). We propose that the NOD-like receptor protein-3 (NLRP3) inflammasome, which has been linked to inflammatory responses in models of ischemic stroke and various inflammatory diseases, may be one mechanism by which connexin hemichannel opening especially mediates perinatal brain injury.

The inflammasome pathway is amplified and perpetuated in an autocrine manner through connexin43 hemichannel mediated ATP release

A HIF-1 Target, ATIA, Protects Cells from Apoptosis by Modulating the Mitochondrial Thioredoxin, TRX2

Connexin hemichannels are single membrane channels which have been traditionally thought to work in pairs to form gap junction channels across two opposing cells. In astrocytes, gap junction channels allow direct intercellular communication and greatly facilitate the transmission of signals. Recently, there has been growing evidence demonstrating that connexin hemichannels, as well as pannexin channels, on their own are open in various conditions. They allow bidirectional flow of ions and signaling molecules and act as release sites for transmitters like ATP and glutamate into the extracellular space. While much attention has focused on the function of connexin hemichannels and pannexons during pathological situations like epilepsy, inflammation, neurodegeneration or ischemia, their potential roles in physiology is often ignored. In order to fully understand the dynamic properties and roles of connexin hemichannels and pannexons in the brain, it is essential to decipher whether they also have some physiological functions and contribute to normal cerebral processes. Here, we present recent studies in the CNS suggesting emerging physiological functions of connexin hemichannels and pannexons in normal neuronal activity and behavior. We also discuss how these pioneer studies pave the way for future research to extend the physiological relevance of connexons and pannexons, and some fundamental issues yet to be addressed.

Hemichannels are transmembrane channels that represent the functional subunits of gap junctions. Each hemichannel is composed of a connexin or pannexin hexamer and, after being transported to the membrane, remains unpaired until it is incorporated in a gap junction. Several studies have already provided evidence that gap junction-mediated intercellular diffusion of ions and small molecules during ischemia represents an important mechanism through which necrotic, apoptotic, or even protective signals are transported between cells. Although initially hemichannels were supposed to be functional only in gap junctions, recent findings indicate that unpaired hemichannels also display a large array of activities that can be modulated under several pathophysiological conditions, including ischemia. Open hemichannels in ischemia dramatically alter the permeability properties of membranes and lead to cell death through ionic dysregulation, loss of metabolites, and changes in intracellular ATP. This review focuses on the properties and possible functions of unpaired connexin and pannexin hemichannels and the implications this has for a variety of events, such as cell death, glutamate release, oxidative stress, cortical spreading depression, that occur during an ischemic insult and may affect its outcome.

Abnormally increased activity of connexin hemichannels contributes to cell damage in many disorders, including deafness, stroke, and cardiac infarct, and therefore hemichannels constitute a potentially important therapeutic target. Unfortunately, the available hemichannel inhibitors are not specific and most are toxic. The absence of a simple and cost-effective screening assay has made the discovery of hemichannel inhibitors difficult. Here, we present an optimized assay where human connexins are expressed in genetically modified Escherichia coli cells deficient in potassium uptake (LB2003 cells). These cells cannot grow in low-potassium medium, and hemichannel function is assayed by the reversion of the no-growth phenotype. Since functional hemichannels are permeable to potassium, they allow for its uptake and cell growth. The simple reading of bacterial growth in low-potassium medium distinguishes functional hemichannels (growth) from those inhibited (no growth). This assay is simple, robust, inexpensive, and reliable, and is easily scaled to high-throughput multiwell platforms. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of competent LB2003 cells resistant to kanamycin Basic Protocol 2: Growth complementation assay Support Protocol: Evaluation of cytotoxic effects of potential connexin hemichannel inhibitors.

Connexin43 hemichannel block inhibits NLRP3 inflammasome activation in a human retinal explant model of diabetic retinopathy

Perinatal encephalopathy remains a major cause of disability, such as cerebral palsy. Therapeutic hypothermia is now well established to partially reduce risk of disability in late preterm/term infants. However, new and complementary therapeutic targets are needed to further improve outcomes. There is increasing evidence that glia play a key role in neural damage after hypoxia-ischemia and infection/inflammation. In this review, we discuss the role of astrocytic gap junction (connexin) hemichannels in the spread of neural injury after hypoxia-ischemia and/or infection/inflammation. Potential mechanisms of hemichannel mediated injury likely involve impaired intracellular calcium handling, loss of blood-brain barrier integrity and release of adenosine triphosphate (ATP) resulting in over-activation of purinergic receptors. We propose the hypothesis that inflammation-induced opening of connexin hemichannels is a key regulating event that initiates a vicious cycle of excessive ATP release, which in turn propagates activation of purinergic receptors on microglia and astrocytes. This suggests that developing new neuroprotective strategies for preterm infants will benefit from a detailed understanding of glial and connexin hemichannel responses.