Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^?/? mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^?/? mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Intrinsic properties and regulation of Pannexin 1 channel

Pannexin 1 (Panx1) channels are generally represented as non-selective, large-pore channels that release ATP. Emerging roles have been described for Panx1 in mediating purinergic signaling in the normal nervous, cardiovascular, and immune systems, where they may be activated by mechanical stress, ionotropic and metabotropic receptor signaling, and via proteolytic cleavage of the Panx1 C-terminus. Panx1 channels are widely expressed in various cell types, and it is now thought that targeting these channels therapeutically may be beneficial in a number of pathophysiological contexts, such as asthma, atherosclerosis, hypertension, and ischemic-induced seizures. Even as interest in Panx1 channels is burgeoning, some of their basic properties, mechanisms of modulation, and proposed functions remain controversial, with recent reports challenging some long-held views regarding Panx1 channels. In this brief review, we summarize some well-established features of Panx1 channels; we then address some current confounding issues surrounding Panx1 channels, especially with respect to intrinsic channel properties, in order to raise awareness of these unsettled issues for future research.     

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^−/− mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^−/− mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Channel activity of recombinant pannexin 1

Mammalian taste cells of the type II release ATP, an afferent neurotransmitter, by employing unselective ATP-permeable ion channels. The molecular identity of these channels is not known with confidence, although evidence implicates certain channel proteins from the connexin and pannexin families as most likely candidates. Here we carried out the comparative analysis of biophysical features and pharmacological profiles of unselective channels operative in type II cells and recombinant pannexin 1 (Panx1), which was cloned from the taste tissue and heterologously expressed in eukaryotic cells of several lines, including HEK-293, CHO, and neuroblastoma SK-N-SH. Integral currents mediated by Panx1 hemichannels were recorded to elucidate their kinetics characteristics, such as activation and deactivation, voltage dependence, and sensitivity to a variety of blockers, including carbenoxolone, DIDS, and NPPB. It was shown that the heterologous expression of Panx1 in cells of each type induced specific conductance, which exhibited outward rectification and was effectively blockable with carbenoxolone and anionic channel blockers DIDS and NPPB. Panx1 activity was studied at the single channel level as well. As was found, transfection of HEK-293 cells with the plasmid harboring cDNA encoding Panx1 gave rise to single channel current-like events in excised patches that were inhibited by 20 μM carbenoxolone, the relatively specific blocker of Panx1. These carbenoxolone-sensitive channels were peculiar in that single-channel current versus membrane voltage was not linear but exhibited outward rectification. In addition, the open-channel probability strongly increased with membrane voltage. Taken together, the data obtained here and earlier demonstrate clearly that by their biophysical and pharmacological features, ATP-permeable channels operative in type II cells are rather distinct from recombinant Panx1 hemichannels, thus arguing against Panx1 as the main conduit of ATP release in taste cells.     

Pannexin 1 as a driver of inflammation and ischemia–reperfusion injury

Pannexin 1 (Panx1) is a ubiquitously expressed protein forming large conductance channels that are central to many distinct inflammation and injury responses. There is accumulating evidence showing ATP released from Panx1 channels, as well as metabolites, provide effective paracrine and autocrine signaling molecules that regulate different elements of the injury response. As channels with a broad range of permselectivity, Panx1 channels mediate the secretion and uptake of multiple solutes, ranging from calcium to bacterial derived molecules. In this review, we describe how Panx1 functions in response to different pro-inflammatory stimuli, focusing mainly on signaling coordinated by the vasculature. How Panx1 mediates ATP release by injured cells is also discussed. The ability of Panx1 to serve as a central component of many diverse physiologic responses has proven to be critically dependent on the context of expression, post-translational modification, interacting partners, and the mode of stimulation.     

Pannexin1-Mediated ATP Release Provides Signal Transmission Between Neuro2A Cells

Pannexin1 (Panx1), a protein related to the gap junction proteins of invertebrates, forms nonjunctional channels that open upon depolarization and in response to mechanical stretch and purinergic receptor stimulation. Importantly, ATP can be released through Panx1 channels, providing a possible role for these channels in non-vesicular signal transmission. In this study we expressed exogenous human and mouse Panx1 in the gap junction deficient Neuro2A neuroblastoma cell line and explored the contribution of Panx1 channels to cell–cell communication as sites of ATP release. Electrophysiological (patch clamp) recordings from Panx1 transfected Neuro2A cells revealed membrane conductance that increased beyond 0 mV when applying voltage ramps from −60 to +100 mV; threshold was correlated with extracellular K⁺, so that at 10 mM K⁺, channels began to open at −30 mV. Evaluation of cell–cell communication using dual whole cell recordings from cell pairs revealed that activation of Panx1 current in one cell of the pair induced an inward current in the second cell after a latency of 10–20 s. This paracrine response was amplified by an ATPase inhibitor (ARL67156, 100 μM) and was blocked by the ATP-degrading enzyme apyrase (6.7 U/ml), by the P2 receptor antagonist suramin (50 μM) and by the Panx1 channel blocker carbenoxolone. These results provide additional evidence that ATP release through Panx1 channels can mediate nonsynaptic bidirectional intercellular communication. Furthermore, current potentiation by elevated K⁺ provides a mechanism for enhancement of ATP release under pathological conditions.     

Chemotherapeutic Drugs Induce ATP Release via Caspase-gated Pannexin-1 Channels and a Caspase/Pannexin-1-independent Mechanism

Anti-tumor immune responses have been linked to the regulated release of ATP from apoptotic cancer cells to engage P2 purinergic receptor signaling cascades in nearby leukocytes. We used the Jurkat T cell acute lymphocytic leukemia model to characterize the role of pannexin-1 (Panx1) channels in the release of nucleotides during chemotherapeutic drug-induced apoptosis. Diverse pro-apoptotic drugs, including topoisomerase II inhibitors, kinase inhibitors, and proteosome inhibitors, induced functional activation of Panx1 channels via caspase-3-mediated cleavage of the Panx1 autoinhibitory C-terminal domain. The caspase-activated Panx1 channels mediated efflux of ATP, but also ADP and AMP, with the latter two comprising >90% of the released adenine nucleotide pool as cells transitioned from the early to late stages of apoptosis. Chemotherapeutic drugs also activated an alternative caspase- and Panx1-independent pathway for ATP release from Jurkat cells in the presence of benzyloxycarbonyl-VAD, a pan-caspase inhibitor. Comparison of Panx1 levels indicated much higher expression in leukemic T lymphocytes than in normal, untransformed T lymphoblasts. This suggests that signaling roles for Panx1 may be amplified in leukemic leukocytes. Together, these results identify chemotherapy-activated pannexin-1 channels and ATP release as possible mediators of paracrine interaction between dying tumor cells and the effector leukocytes that mediate immunogenic anti-tumor responses.     

Two non-vesicular ATP release pathways in the mouse erythrocyte membrane

Erythrocytes are exceptionally suited for analysis of non-exocytotic release mechanisms of ATP, because these cells under physiological conditions lack vesicles. Previous studies have indicated, that Pannexin1 (Panx1) provides a key ATP permeation pathway in many cell types, including human and frog erythrocytes. Here we show that erythrocytes of Panx1(-/-) mice lend further support to this conclusion. However, ATP release, although attenuated, was still observed in Panx1(-/-) mouse erythrocytes. In contrast to Panx1(+/+) cells, this release was not correlated with uptake of extracellularly applied dyes, was insensitive to Panx1 channel blockers, and was inhibited by dipyridamole and stimulated by iloprost. Thus, in erythrocytes, two independent pathways mediate the release of ATP. We also show that glyburide is a strong inhibitor of Panx1 channels.     

Carbenoxolone inhibits Pannexin1 channels through interactions in the first extracellular loop

Pannexin1 (Panx1) is an ATP release channel important for controlling immune responses and synaptic strength. Various stimuli including C-terminal cleavage, a high concentration of extracellular potassium, and voltage have been demonstrated to activate Panx1. However, it remains unclear how Panx1 senses and integrates such diverse stimuli to form an open channel. To provide a clue on the mechanism underlying Panx1 channel gating, we investigated the action mechanism of carbenoxolone (CBX), the most commonly used small molecule for attenuating Panx1 function triggered by a wide range of stimuli. Using a chimeric approach, we discovered that CBX reverses its action polarity and potentiates the voltage-gated channel activity of Panx1 when W74 in the first extracellular loop is mutated to a nonaromatic residue. A systematic mutagenesis study revealed that conserved residues in this loop also play important roles in CBX function, potentially by mediating CBX binding. We extended our experiments to other Panx1 inhibitors such as probenecid and ATP, which also potentiate the voltage-gated channel activity of a Panx1 mutant at position 74. Notably, probenecid alone can activate this mutant at a resting membrane potential. These data suggest that CBX and other inhibitors, including probenecid, attenuate Panx1 channel activity through modulation of the first extracellular loop. Our experiments are the first step toward identifying a previously unknown mode of CBX action, which provide insight into the role of the first extracellular loop in Panx1 channel gating.     

The role of pannexin1 in the induction and resolution of inflammation

Extracellular ATP is an important signaling molecule throughout the inflammatory cascade, serving as a danger signal that causes activation of the inflammasome, enhancement of immune cell infiltration, and fine-tuning of several signaling cascades including those important for the resolution of inflammation. Recent studies demonstrated that ATP can be released from cells in a controlled manner through pannexin (Panx) channels. Panx1-mediated ATP release is involved in inflammasome activation and neutrophil/macrophage chemotaxis, activation of T cells, and a role for Panx1 in inducing and propagating inflammation has been demonstrated in various organs, including lung and the central and peripheral nervous system. The recognition and clearance of dying cells and debris from focal points of inflammation is critical in the resolution of inflammation, and Panx1-mediated ATP release from dying cells has been shown to recruit phagocytes. Moreover, extracellular ATP can be broken down by ectonucleotidases into ADP, AMP, and adenosine, which is critical in the resolution of inflammation. Together, Panx1, ATP, purinergic receptors, and ectonucleotidases contribute to important feedback loops during the inflammatory response, and thus represent promising candidates for new therapies.     

Contribution of Pannexin1 to Experimental Autoimmune Encephalomyelitis

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.     

Pannexin1 drives multicellular aggregate compaction via a signaling cascade that remodels the actin cytoskeleton

Pannexin 1 (Panx1) is a novel gap junction protein shown to have tumor-suppressive properties. To model its in vivo role in the intratumor biomechanical environment, we investigated whether Panx1 channels modulate the dynamic assembly of multicellular C6 glioma aggregates. Treatment with carbenoxolone and probenecid, which directly and specifically block Panx1 channels, respectively, showed that Panx1 is involved in accelerating aggregate assembly. Experiments further showed that exogenous ATP can reverse the inhibitive effects of carbenoxolone and that aggregate compaction is sensitive to the purinergic antagonist suramin. With a close examination of the F-actin microfilament network, these findings show that Panx1 channels act as conduits for ATP release that stimulate the P(2)X(7) purinergic receptor pathway, in turn up-regulating actomyosin function. Using a unique three-dimensional scaffold-free method to quantify multicellular interactions, this study shows that Panx1 is intimately involved in regulating intercellular biomechanical interactions pivotal in the progression of cancer.     

Pannexin1 Channel Proteins in the Zebrafish Retina Have Shared and Unique Properties

In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.     

Interactions of Pannexin1 channels with purinergic and NMDA receptor channels

Pannexins are a three-member family of vertebrate plasma membrane spanning molecules that have homology to the invertebrate gap junction forming proteins, the innexins. However, pannexins do not form gap junctions but operate as plasma membrane channels. The best-characterized member of these proteins, Pannexin1 (Panx1) was suggested to be functionally associated with purinergic P2X and N-methyl-D-aspartate (NMDA) receptor channels. Activation of these receptor channels by their endogenous ligands leads to cross-activation of Panx1 channels. This in turn potentiates P2X and NMDA receptor channel signaling. Two potentiation concepts have been suggested: enhancement of the current responses and/or sustained receptor channel activation by ATP released through Panx1 pore and adenosine generated by ectonucleotidase-dependent dephosphorylation of ATP. Here we summarize the current knowledge and hypotheses about interactions of Panx1 channels with P2X and NMDA receptor channels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Thrombin-induced ATP release from human umbilical vein endothelial cells

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.     

Inhibition of pannexin-1 channel activity by adiponectin in podocytes: Role of acid ceramidase activation

The pannexin-1 (Panx1) channel has been reported to mediate the release of ATP that is involved in local tissue inflammation, obesity, and many chronic degenerative diseases. It remains unknown whether Panx1 is present in podocytes and whether this channel in podocytes mediates ATP release leading to glomerular inflammation or fibrosis. To answer these questions, we first characterized the expression of Panx channels in podocytes. Among the three known pannexins, Panx1 was the most enriched in podocytes, either cultured or native in mouse glomeruli. Using a Port-a-Patch planar patch-clamp system, we recorded a large voltage-gated outward current through podocyte membrane under the Cs⁺in/Na⁺out gradient. Substitution of gluconate or aspartate for chloride in the bath solution blocked voltage-gated outward currents and shifted the reversal potential of Panx1 currents to the right, indicating the anion permeability of this channel. Pharmacologically, the recorded voltage-gated outward currents were substantially attenuated by specific Panx1 channel inhibitors. Given the anti-inflammatory and intracellular ATP restorative effects of adiponectin, we tested whether this adipokine inhibits Panx1 channel activity to block ATP release. Adiponectin blocked Panx1 channel activity in podocytes. Mechanistically, inhibition of acid ceramidase (AC) remarkably enhanced Panx1 channel activity under control conditions and prevented the inhibition of Panx1 channel by adiponectin. Correspondingly, intracellular addition of AC products, sphingosine or sphingosine-1-phosphate (S1P), blocked Panx1 channel activity, while elevation of intracellular ceramide had no effect on Panx1 channel activity. These results suggest that adiponectin inhibits Panx1 channel activity in podocytes through activation of AC and associated elevation of intracellular S1P.