Pannexin Hemichannels Research Articles

Pathophysiological role of connexin and pannexin hemichannels in neuromuscular disorders.

A growing body of research has provided evidence that de novo expression of connexin hemichannels and upregulation of pannexin hemichannels (Cx HCs and Panx HCs, respectively) in the cytoplasmic membrane of skeletal muscle (sarcolemma) are critical steps in the pathogenesis of muscle dysfunction of many genetic and acquired muscle diseases. This review provides an overview of the current understanding of the molecular mechanisms regulating the expression of Cx and Panx HCs in skeletal muscle, as well as their roles in both muscle physiology and pathologies. Additionally, it addresses existing gaps in knowledge and outlines future challenges in the field.

Ca2+ permeation through C-terminal cleaved, but not full-length human Pannexin1 hemichannels, mediates cell death

Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd+, but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca2+ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca2+ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca2+ failed to induce cell death, and the Ca2+ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or 10Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1-/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca2+ overload that promotes cell death.

Glial Cx43 hemichannels and neuronal Panx1 hemichannels and P2X7 receptors orchestrate presynaptic homeostatic plasticity

The emerging role of glial cells in modulating neuronal excitability and synaptic strength is a growing field in neuroscience. In recent years, a pivotal role of gliotransmission in homeostatic presynaptic plasticity has been highlighted and glial-derived ATP arises as a key contributor. However, very little is known about the glial non-vesicular ATP-release pathway and how ATP participates in the modulation of synaptic strength. Here, we investigated the functional changes occurring in neurons upon chronic inactivity and the role of the purinergic signaling, connexin43 and pannexin1 hemichannels in this process. By using hippocampal dissociated cultures, we showed that blocking connexin43 and pannexin1 hemichannels decreases the amount of extracellular ATP. Moreover, Ca2+ imaging assays using Fluo-4/AM revealed that blocking connexin43, neuronal P2X7Rs and pannexin1 hemichannels decreases the amount of basal Ca2+ in neurons. A significant impairment in synaptic vesicle pool size was also evidenced under these conditions. Interestingly, rescue experiments where Panx1HCs are blocked showed that the compensatory adjustment of cytosolic Ca2+ was recovered after P2X7Rs activation, suggesting that Panx1 acts downstream P2X7Rs. These changes were accompanied by a modulation of neuronal permeability, as revealed by ethidium bromide uptake experiments. In particular, the permeability of neuronal P2X7Rs and pannexin1 hemichannels is increased upon 24 h of inactivity. Taken together, we have uncovered a role for connexin43-dependent ATP release and neuronal P2X7Rs and pannexin1 hemichannels in the adjustment of presynaptic strength by modulating neuronal permeability, the entrance of Ca2+ into neurons and the size of the recycling pool of synaptic vesicles.

Acute activation of hemichannels by ethanol leads to Ca2+-dependent gliotransmitter release in astrocytes.

Multiple studies have demonstrated that acute ethanol consumption alters brain function and cognition. Nevertheless, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity, and recently, the opening of hemichannels has been found to play a relevant role in this process. Hemichannels are plasma membrane channels composed of six connexins or seven pannexins, respectively, that oligomerize around a central pore. They serve as ionic and molecular exchange conduits between the cytoplasm and extracellular milieu, allowing the release of various paracrine substances, such as ATP, D-serine, and glutamate, and the entry of ions and other substances, such as Ca2+ and glucose. The persistent and exacerbated opening of hemichannels has been associated with the pathogenesis and progression of several brain diseases for at least three mechanisms. The uncontrolled activity of these channels could favor the collapse of ionic gradients and osmotic balance, the release of toxic levels of ATP or glutamate, cell swelling and plasma membrane breakdown and intracellular Ca2+ overload. Here, we evaluated whether acute ethanol exposure affects the activity of astrocyte hemichannels and the possible repercussions of this phenomenon on cytoplasmatic Ca2+ signaling and gliotransmitter release. Acute ethanol exposure triggered the rapid activation of connexin43 and pannexin1 hemichannels in astrocytes, as measured by time-lapse recordings of ethidium uptake. This heightened activity derived from a rapid rise in [Ca2+]i linked to extracellular Ca2+ influx and IP3-evoked Ca2+ release from intracellular Ca2+ stores. Relevantly, the acute ethanol-induced activation of hemichannels contributed to a persistent secondary increase in [Ca2+]i. The [Ca2+]i-dependent activation of hemichannels elicited by ethanol caused the increased release of ATP and glutamate in astroglial cultures and brain slices. Our findings offer fresh perspectives on the potential mechanisms behind acute alcohol-induced brain abnormalities and propose targeting connexin43 and pannexin1 hemichannels in astrocytes as a promising avenue to prevent deleterious consequences of alcohol consumption.

Hemichannels contribute to mitochondrial Ca2+ and morphology alterations evoked by ethanol in astrocytes.

Alcohol, a toxic and psychoactive substance with addictive properties, severely impacts life quality, leading to significant health, societal, and economic consequences. Its rapid passage across the blood-brain barrier directly affects different brain cells, including astrocytes. Our recent findings revealed the involvement of pannexin-1 (Panx1) and connexin-43 (Cx43) hemichannels in ethanol-induced astrocyte dysfunction and death. However, whether ethanol influences mitochondrial function and morphology in astrocytes, and the potential role of hemichannels in this process remains poorly understood. Here, we found that ethanol reduced basal mitochondrial Ca2+ but exacerbated thapsigargin-induced mitochondrial Ca2+ dynamics in a concentration-dependent manner, as evidenced by Rhod-2 time-lapse recordings. Similarly, ethanol-treated astrocytes displayed increased mitochondrial superoxide production, as indicated by MitoSox labeling. These effects coincided with reduced mitochondrial membrane potential and increased mitochondrial fragmentation, as determined by MitoRed CMXRos and MitoGreen quantification, respectively. Crucially, inhibiting both Cx43 and Panx1 hemichannels effectively prevented all ethanol-induced mitochondrial abnormalities in astrocytes. We speculate that exacerbated hemichannel activity evoked by ethanol may impair intracellular Ca2+ homeostasis, stressing mitochondrial Ca2+ with potentially damaging consequences for mitochondrial fusion and fission dynamics and astroglial bioenergetics.

P2X7 receptors and pannexin1 hemichannels shape presynaptic transmission.

Over the last decades, since the discovery of ATP as a transmitter, accumulating evidence has been reported about the role of this nucleotide and purinergic receptors, in particular P2X7 receptors, in the modulation of synaptic strength and plasticity. Purinergic signaling has emerged as a crucial player in orchestrating the molecular interaction between the components of the tripartite synapse, and much progress has been made in how this neuron-glia interaction impacts neuronal physiology under basal and pathological conditions. On the other hand, pannexin1 hemichannels, which are functionally linked to P2X7 receptors, have appeared more recently as important modulators of excitatory synaptic function and plasticity under diverse contexts. In this review, we will discuss the contribution of ATP, P2X7 receptors, and pannexin hemichannels to the modulation of presynaptic strength and its impact on motor function, sensory processing, synaptic plasticity, and neuroglial communication, with special focus on the P2X7 receptor/pannexin hemichannel interplay. We also address major hypotheses about the role of this interaction in physiological and pathological circumstances.

Antiproliferative effect of boldine on neural progenitor cells and on glioblastoma cells.

The subventricular zone (SVZ) is a brain region that contains neural stem cells and progenitor cells (NSCs/NPCs) from which new neurons and glial cells are formed during adulthood in mammals. Recent data indicate that SVZ NSCs are the cell type that acquires the initial tumorigenic mutation in glioblastoma (GBM), the most aggressive form of malignant glioma. NSCs/NPCs of the SVZ present hemichannel activity whose function has not yet been fully elucidated. In this work, we aimed to analyze whether hemichannel-mediated communication affects proliferation of SVZ NPCs and GBM cells. For that purpose, we used boldine, an alkaloid derived from the boldo tree (Peumus boldus), that inhibits connexin and pannexin hemichannels, but without affecting gap junctional communication. Boldine treatment (50 μM) of rat SVZ NPCs grown as neurospheres effectively inhibited dye uptake through hemichannels and induced a significant reduction in neurosphere diameter and in bromodeoxyuridine (BrdU) incorporation. However, the differentiation pattern was not modified by the treatment. Experiments with specific blockers for hemichannels formed by connexin subunits (D4) or pannexin 1 (probenecid) revealed that probenecid, but not D4, produced a decrease in BrdU incorporation similar to that obtained with boldine. These results suggest that inhibition of pannexin 1 hemichannels could be partially responsible for the antiproliferative effect of boldine on SVZ NPCs. Analysis of the effect of boldine (25-600 μM) on different types of primary human GBM cells (GBM59, GBM96, and U87-MG) showed a concentration-dependent decrease in GBM cell growth. Boldine treatment also induced a significant inhibition of hemichannel activity in GBM cells. Altogether, we provide evidence of an antimitotic action of boldine in SVZ NPCs and in GBM cells which may be due, at least in part, to its hemichannel blocking function. These results could be of relevance for future possible strategies in GBM aimed to suppress the proliferation of mutated NSCs or glioma stem cells that might remain in the brain after tumor resection.

Purinergic 2X 4 (P2X4), but not P2X7, receptors increase cytosolic [Ca2+] and stimulate mucin secretion in rat conjunctival goblet cells to maintain ocular surface health

Ionotropic purinergic receptors (P2XRs) are activated by ATP and ATP analogs. ATP can be released through ATP-permeable channels such as the pannexin hemichannels. Upon activation, the P2XRs become permeable to Ca2+, a potent stimulator of mucin secretion in conjunctival goblet cells (CGCs). The purpose of this study was to investigate the presence and function of P2XRs in CGCs. We also examined the presence of pannexin hemichannels. Rat first passage CGCs were stained with the goblet cell marker anti-cytokeratin 7 antibody and specific antibodies to P2X1-7 receptors and pannexin 1-3. mRNA expression was determined by RT-PCR using primers specific to P2XRs and pannexins. Proteins were identified with Western blotting (WB) using the same antibodies as for immunofluorescence (IF) microscopy. To study receptor function, CGCs were incubated with Fura 2-AM, exposed to agonists and antagonists, and intracellular [Ca2+] ([Ca2+]i) measured. [Ca2+]i was also measured after knock down of P2X4 and P2X7 receptor expression, and when exploiting P2XR specific characteristics. Lastly, mucin secretion was measured after the addition of several P2XR agonists. All P2XRs and pannexins were visualized with IF microscopy, and identified with RT-PCR and WB. [Ca2+]i was significantly increased when stimulated with ATP (10−7-10−4 M). Suramin, a non-selective P2XR antagonist at 10−4 M did not reduce ATP-induced peak [Ca2+]i. The potent P2X7 agonist, BzATP (10−7-10−4 M) increased the [Ca2+]i, although to a lesser extent than ATP. When measuring [Ca2+]i the effect of repeated applications of ATP at 10−5 or 10−6 M the response “desensitized” after 30–60 s. The P2X4 specific antagonist 5-BDBD decreased the P2X4 agonist, 2MeSATP,-induced [Ca2+]i increase. Furthermore, siRNA against the P2X4R, but not the P2X7R, decreased agonist-induced peak [Ca2+]i. ATP (10−5 M), BzATP (10−4 M) and 2MeSATP (10−5 M) induced mucin secretion. We conclude that all seven P2XRs are present in cultured rat CGCs. Of the P2XRs, only activation of the homotrimeric P2X4R appears to increase [Ca2+]i and induce mucin secretion. The P2X4R in CGCs offers a new therapeutic target for protective mucin secretion.

Pannexin-1 Modulates Inhibitory Transmission and Hippocampal Synaptic Plasticity.

Pannexin-1 (Panx1) hemichannel is a non-selective transmembrane channel that may play important roles in intercellular signaling by allowing the permeation of ions and metabolites, such as ATP. Although recent evidence shows that the Panx1 hemichannel is involved in controlling excitatory synaptic transmission, the role of Panx1 in inhibitory transmission remains unknown. Here, we studied the contribution of Panx1 to the GABAergic synaptic efficacy onto CA1 pyramidal neurons (PyNs) by using patch-clamp recordings and pharmacological approaches in wild-type and Panx1 knock-out (Panx1-KO) mice. We reported that blockage of the Panx1 hemichannel with the mimetic peptide 10Panx1 increases the synaptic level of endocannabinoids (eCB) and the activation of cannabinoid receptors type 1 (CB1Rs), which results in a decrease in hippocampal GABAergic efficacy, shifting excitation/inhibition (E/I) balance toward excitation and facilitating the induction of long-term potentiation. Our finding provides important insight unveiling that Panx1 can strongly influence the overall neuronal excitability and play a key role in shaping synaptic changes affecting the amplitude and direction of plasticity, as well as learning and memory processes.

Sirtuin 6 Regulates the Activation of the ATP/Purinergic Axis in Endothelial Cells.

Sirtuin 6 (SIRT6) is a member of the mammalian NAD+-dependent deac(et)ylase sirtuin family. SIRT6's anti-inflammatory roles are emerging increasingly often in different diseases and cell types, including endothelial cells. In this study, the role of SIRT6 in pro-inflammatory conditions was investigated by engineering human umbilical vein endothelial cells to overexpress SIRT6 (SIRT6+ HUVECs). Our results showed that SIRT6 overexpression affected the levels of adhesion molecules and sustained megakaryocyte proliferation and proplatelet formation. Interestingly, the pro-inflammatory activation of the ATP/purinergic axis was reduced in SIRT6+ HUVECs. Specifically, the TNFα-induced release of ATP in the extracellular space and the increase in pannexin-1 hemichannel expression, which mediates ATP efflux, were hampered in SIRT6+ cells. Instead, NAD+ release and Connexin43 expression were not modified by SIRT6 levels. Moreover, the Ca2+ influx in response to ATP and the expression of the purinergic receptor P2X7 were decreased in SIRT6+ HUVECs. Contrary to extracellular ATP, extracellular NAD+ did not evoke pro-inflammatory responses in HUVECs. Instead, NAD+ administration reduced endothelial cell proliferation and motility and counteracted the TNFα-induced angiogenesis. Altogether, our data reinforce the view of SIRT6 activation as an anti-inflammatory approach in vascular endothelium.

TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation.

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.

β1 Integrin induces adhesion and migration of human Th17 cells via Pyk2-dependent activation of P2X4 receptor.

Integrin-mediated T-cell adhesion and migration is a crucial step in immune response and autoimmune diseases. However, the underlying signalling mechanisms are not fully elucidated. In this study, we examined the implication of purinergic signalling, which has been associated with T-cell activation, in the adhesion and migration of human Th17 cells across fibronectin, a major matrix protein associated with inflammatory diseases. We showed that the adhesion of human Th17 cells to fibronectin induces, via β1 integrin, a sustained release of adenosine triphosphate (ATP) from the mitochondria through the pannexin-1 hemichannels. Inhibition of ATP release or its degradation with apyrase impaired the capacity of the cells to attach and migrate across fibronectin. Inhibition studies identified a major role for the purinergic receptor P2X4 in T-cell adhesion and migration but not for P2X7 or P2Y11 receptors. Blockade of P2X4 but not P2X7 or P2Y11 receptors reduced cell adhesion and migration by inhibiting activation of β1 integrins, which is essential for ligand binding. Furthermore, we found that β1 integrin-induced ATP release, P2X4 receptor transactivation, cell adhesion and migration were dependent on the focal adhesion kinase Pyk2 but not FAK. Finally, P2X4 receptor inhibition also blocked fibronectin-induced Pyk2 activation suggesting the existence of a positive feedback loop of activation between β1 integrin/Pyk2 and P2X4 purinergic signalling pathways. Our findings uncovered an unrecognized link between β1 integrin and P2X4 receptor signalling pathways for promoting T-cell adhesion and migration across the extracellular matrix.

Single channel properties of pannexin-1 and connexin-43 hemichannels and P2X7 receptors in astrocytes cultured from rodent spinal cords.

Astrocytes express surface channels involved in purinergic signaling. Among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) release ATP that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, that is, open and close under physiological and pathological conditions, single channel properties of Px1 HCs in astrocytes have not been defined. Here, we developed a dual voltage clamp technique in HeLa cells expressing human Px1-YFP, and then applied this system to rodent spinal astrocytes to compare their single channel properties with other surface channels, that is, Cx43 HCs and P2X7 receptors (P2X7Rs). Channels were recorded in cell attached patches and evoked with ramp cycles applied through another pipette in whole cell voltage clamp. The mean unitary conductances of Px1 HCs were comparable in HeLa Px1-YFP cells and spinal astrocytes, ~42 and ~48 pS, respectively. Based on their unitary conductance, voltage-dependence, and unitary activity after pharmacological and gene silencing, Px1 HCs in astrocytes could be distinguished from Cx43 HCs and P2X7Rs. Channel activity of Px1 HCs and P2X7Rs was greater than that of Cx43 HCs in control astrocytes during ramps. Unitary activity of Px1 HCs was decreased and that of Cx43 HCs and P2X7Rs increased in astrocytes treated with fibroblast growth factor 1 (FGF-1). In summary, we resolved single channel properties of three different surface channels involved in purinergic signaling in spinal astrocytes, which were differentially modulated by FGF-1, a growth factor involved in neurodevelopment, inflammation and repair.

Characterization of Pannexin1, Connexin32, and Connexin43 in Spotted Sea Bass (Lateolabrax maculatus): They Are Important Neuro-Related Immune Response Genes Involved in Inflammation-Induced ATP Release.

Many immunological diseases can be treated by regulating neurobehavior, in which extracellular ATP is a vital member of endogenous danger-associated molecular pattern signaling molecule that plays a crucial part in innate neuro-related immunity. It is actively released through pannexin (Panx) and connexin (Cx) hemichannels from activated or stressed cells during inflammation, injury, or apoptosis. In addition to participating in ATP release, Panxs and Cxs also have crucial immune functions. In this study, pannexin1, three connexin32 isoforms and connexin43 were identified and characterized in spotted sea bass (Lateolabrax maculatus), which were named LmPanx1, LmCx32.2, LmCx32.3, LmCx32.7, and LmCx43. Their similar topological structures were discovered by sequence analysis: a relatively unconserved C-terminal region and four highly conserved transmembrane (TM) domains, and so on. Each extracellular (ECL) region of Panx1 has two conserved cysteine residues. Unlike Panx1, each ECL region of Cx32 and Cx43 contains three conserved cysteine residues, forming two conserved motifs: CX6CX3C motif in ECL1 and CX4CX5C motif in ECL2. Furthermore, Panx1 and Cx43 share similar genomic organization and synteny with their counterparts in selected vertebrates. Cx32 and CX43 were located in the same locus in fish, but diverged into two loci from amphibian. Moreover, despite varying expression levels, the identified genes were constitutively expressed in all examined tissues. All genes were upregulated by PAMP [lipopolysaccharide and poly(I:C)] stimulation or bacterial infection in vivo and in vitro, but they were downregulated in the brain at 6 or 12 h after stimulation. Especially, the three LmCx32 isoforms and LmCx43 were upregulated by ATP stimulation in primary head kidney leukocytes; however, downregulation of LmCx32.3 and LmCx43 expression were noted at 12 h. Conversely, ATP treatment inhibited the expression of LmPanx1. Importantly, we showed that the spotted sea bass Panx1, Cx43, and Cx32 were localized on the cellular membrane and involved in inflammation-induced ATP release. Taken together, our results demonstrated that Panx1, Cx32, and Cx43 are important neuro-related immune response genes involved in inflammation-induced ATP release.

Apelin-13/APJ induces cardiomyocyte hypertrophy by activating the Pannexin-1/P2X7 axis and FAM134B-dependent reticulophagy.

Cardiac hypertrophy is a leading cause of cardiac morbidity and mortality worldwide. Apelin is the endogenous ligand for the G protein-coupled receptor, APJ. Previously, we have revealed that apelin-13 can induce cardiomyocyte hypertrophy by activating the autophagy pathway. However, the precise mechanism through which apelin-13 regulates reticulophagy to participate in cardiomyocyte hypertrophy remains unclear. Herein, we observed that apelin-13-induced cardiomyocyte hypertrophy by activating FAM134B-dependent reticulophagy via the Pannexin-1/P2X7 signal pathway. Furthermore, we found that apelin-13 stimulated the opening of Pannexin-1 hemichannel and increased extracellular ATP (eATP) levels, which activated the P2X7 purinergic receptor. Activation of the Pannexin-1/eATP/P2X7 axis subsequently led to FAM134B-dependent reticulophagy. Moreover, inhibition of the Pannexin-1/P2X7 axis and FAM134B-dependent reticulophagy reversed apelin-13-induced cardiomyocyte hypertrophy. Based on our present findings, apelin-13/APJ induces cardiomyocyte hypertrophy by activating the Pannexin-1/P2X7 axis and FAM134B-dependent reticulophagy.

Inherent P2X7 Receptors Regulate Macrophage Functions during Inflammatory Diseases.

Macrophages are mononuclear phagocytes which derive either from blood-borne monocytes or reside as resident macrophages in peripheral (Kupffer cells of the liver, marginal zone macrophages of the spleen, alveolar macrophages of the lung) and central tissue (microglia). They occur as M1 (pro-inflammatory; classic) or M2 (anti-inflammatory; alternatively activated) phenotypes. Macrophages possess P2X7 receptors (Rs) which respond to high concentrations of extracellular ATP under pathological conditions by allowing the non-selective fluxes of cations (Na+, Ca2+, K+). Activation of P2X7Rs by still higher concentrations of ATP, especially after repetitive agonist application, leads to the opening of membrane pores permeable to ~900 Da molecules. For this effect an interaction of the P2X7R with a range of other membrane channels (e.g., P2X4R, transient receptor potential A1 [TRPA1], pannexin-1 hemichannel, ANO6 chloride channel) is required. Macrophage-localized P2X7Rs have to be co-activated with the lipopolysaccharide-sensitive toll-like receptor 4 (TLR4) in order to induce the formation of the inflammasome 3 (NLRP3), which then activates the pro-interleukin-1β (pro-IL-1β)-degrading caspase-1 to lead to IL-1β release. Moreover, inflammatory diseases (e.g., rheumatoid arthritis, Crohn’s disease, sepsis, etc.) are generated downstream of the P2X7R-induced upregulation of intracellular second messengers (e.g., phospholipase A2, p38 mitogen-activated kinase, and rho G proteins). In conclusion, P2X7Rs at macrophages appear to be important targets to preserve immune homeostasis with possible therapeutic consequences.

Physical tuning of galectin-3 signaling

Galectin-3 (Gal3) exhibits dynamic oligomerization and promiscuous binding, which can lead to concomitant activation of synergistic, antagonistic, or noncooperative signaling pathways that alter cell behavior. Conferring signaling pathway selectivity through mutations in the Gal3-glycan binding interface is challenged by the abundance of common carbohydrate types found on many membrane glycoproteins. Here, employing alpha-helical coiled-coils as scaffolds to create synthetic Gal3 constructs with defined valency, we demonstrate that oligomerization can physically regulate extracellular signaling activity of Gal3. Constructs with 2 to 6 Gal3 subunits ("Dimer," "Trimer," "Tetramer," "Pentamer," "Hexamer") demonstrated glycan-binding properties and cell death-inducing potency that scaled with valency. Dimer was the minimum functional valency. Unlike wild-type Gal3, which signals apoptosis and mediates agglutination, synthetic Gal3 constructs induced cell death without agglutination. In the presence of CD45, Hexamer was distributed on the cell membrane, whereas it clustered in absence of CD45 via membrane glycans other than those found on CD7. Wild-type Gal3, Pentamer, and Hexamer required CD45 and CD7 to signal apoptosis, and the involvement of caspases in apoptogenic signaling was increased in absence of CD45. However, wild-type Gal3 depended on caspases to signal apoptosis to a greater extent than Hexamer, which had greater caspase dependence than Pentamer. Diminished caspase activation downstream of Hexamer signaling led to decreased pannexin-1 hemichannel opening and interleukin-2 secretion, events facilitated by the increased caspase activation downstream of wild-type Gal3 signaling. Thus, synthetic fixation of Gal3 multivalency can impart physical control of its outside-in signaling activity by governing membrane glycoprotein engagement and, in turn, intracellular pathway activation.

Tuning Multivalent Signaling of Extracellular Galectin‐3

Promiscuous glycan-binding properties of galectin-3 coupled with its dynamic oligomerization can concomitantly activate various signal transduction pathways and, in turn, modulate cell behavior. Modifying the lectin-glycan binding interface to confer signaling pathway selectivity is challenged by the abundance of common carbohydrates that are present on a wide variety of membrane glycoproteins. Alternatively, controlling the multivalent assembly of galectin-3 could provide a physical means to direct its complex extracellular functions. Here, we use a library of α-helical coiled-coils as scaffolds to construct synthetic galectin-3 oligomers with specified valency, and we demonstrate that synthetic galectin-3 assembly can physically regulate membrane glycoprotein recognition to bias the activation of intracellular signaling pathways. Constructs displaying 2-6 galectin-3 subunits (i.e., “Dimer” - “Hexamer”) demonstrated increasing apparent glycan-binding affinity and cell death signaling potency. Though, whereas wild-type galectin-3 signaled caspase-dependent death via CD45 and CD7, Hexamer preferentially signaled death via CD45 due to its abundance and length protruding from the membrane. Preferential engagement of CD45 by Hexamer signaled caspase-independent cell death and diminished caspase-dependent cell activities, such as pannexin-1 hemichannel opening and interleukin-2 secretion, which were more closely associated with wild-type galectin-3 signaling. Therefore, synthetic galectin-3 oligomerization can impart physical control of its carbohydrate-binding promiscuity and, in turn, emergent extracellular functions.

Ectonucleotidases in Acute and Chronic Inflammation.

Ectonucleotidases are extracellular enzymes with a pivotal role in inflammation that hydrolyse extracellular purine and pyrimidine nucleotides, e.g., ATP, UTP, ADP, UDP, AMP and NAD+. Ectonucleotidases, expressed by virtually all cell types, immune cells included, either as plasma membrane-associated or secreted enzymes, are classified into four main families: 1) nucleoside triphosphate diphosphohydrolases (NTPDases), 2) nicotinamide adenine dinucleotide glycohydrolase (NAD glycohydrolase/ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1), 3) ecto-5′-nucleotidase (NT5E), and 4) ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs). Concentration of ATP, UTP and NAD+ can be increased in the extracellular space thanks to un-regulated, e.g., cell damage or cell death, or regulated processes. Regulated processes include secretory exocytosis, connexin or pannexin hemichannels, ATP binding cassette (ABC) transporters, calcium homeostasis modulator (CALMH) channels, the ATP-gated P2X7 receptor, maxi-anion channels (MACs) and volume regulated ion channels (VRACs). Hydrolysis of extracellular purine nucleotides generates adenosine, an important immunosuppressant. Extracellular nucleotides and nucleosides initiate or dampen inflammation via P2 and P1 receptors, respectively. All these agents, depending on their level of expression or activation and on the agonist concentration, are potent modulators of inflammation and key promoters of host defences, immune cells activation, pathogen clearance, tissue repair and regeneration. Thus, their knowledge is of great importance for a full understanding of the pathophysiology of acute and chronic inflammatory diseases. A selection of these pathologies will be briefly discussed here.

Mechanical stretch activates piezo1 in caveolae of alveolar type I cells to trigger ATP release and paracrine stimulation of surfactant secretion from alveolar type II cells.

Secretion of pulmonary surfactant in the alveoli of the lungs is essential to maintain lung function. Stretching of alveoli during lung inflation is the main trigger for surfactant secretion. Yet, the molecular mechanisms how mechanical distension of alveoli results in surfactant secretion are still elusive. The alveolar epithelium consists of alveolar epithelial type I (ATI) and surfactant secreting type II (ATII) cells. ATI, but not ATII cells, express caveolae, small plasma membrane invaginations that can respond to plasma membrane stresses and serve mechanotransductive roles. Within this study, we investigated the role of caveolae as mechanosensors in the alveolus. We generated a human caveolin-1 knockout ATI cell (hAELVicav-/- ) using CRISPR/Cas9. Wildtype (hAELViwt ) and hAELVicav-/- cells grown on flexible membranes responded to increasing stretch amplitudes with rises in intracellular Ca2+ . The response was less frequent and started at higher stretch amplitudes in hAELVicav-/- cells. Stretch-induced Ca2+ -signals depended on Ca2+ -entry via piezo1 channels, localized within caveolae in hAELViwt and primary ATI cells. Ca2+ -entry via piezo1 activated pannexin-1 hemichannels resulting in ATP release from ATI cells. ATP release was reduced in hAELVicav-/- cells. In co-cultures resembling the alveolar epithelium, released ATP stimulated Ca2+ signals and surfactant secretion from neighboring ATII cells when co-cultured with hAELViwt but not hAELVicav-/- cells. In summary, we propose that caveolae in ATI cells are mechanosensors within alveoli regulating stretch-induced surfactant secretion from ATII cells.