Pannexins,distant relatives of the connexin family with specific cellular functions?

Intercellular communication (IC) is mediated by gap junctions (GJs) and hemichannels, which consist of proteins. This has been particularly well documented for the connexin (Cx) family. Initially, Cxs were thought to be the only proteins capable of GJ formation in vertebrates. About 10 years ago, however, a new GJ‐forming protein family related to invertebrate innexins (Inxs) was discovered in vertebrates, and named the pannexin (Panx) family. Panxs, which are structurally similar to Cxs, but evolutionarily distinct, have been shown to be co‐expressed with Cxs in vertebrates. Both protein families show distinct properties and have their own particular function. Identification of the mechanisms that control Panx channel gating is a major challenge for future work. In this review, we focus on the specific properties and role of Panxs in normal and pathological conditions.     

Regulation of connexin‐ and pannexin‐based channels by post‐translational modifications

Connexin (Cx) and pannexin (Panx) proteins form large conductance channels, which function as regulators of communication between neighbouring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signalling, survival and death processes. The functional properties of gap junctions and hemichannels are modulated by different physiological and pathophysiological stimuli. At the molecular level, Cxs and Panxs function as multi‐protein channel complexes, regulating their channel localisation and activity. In addition to this, gap junctional channels and hemichannels are modulated by different post‐translational modifications (PTMs), including phosphorylation, glycosylation, proteolysis, N‐acetylation, S‐nitrosylation, ubiquitination, lipidation, hydroxylation, methylation and deamidation. These PTMs influence almost all aspects of communicating junctional channels in normal cell biology and pathophysiology. In this review, we will provide a systematic overview of PTMs of communicating junction proteins and discuss their effects on Cx and Panx‐channel activity and localisation.     

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.     

Human Pannexin 1 channel: Insight in structure–function mechanism and its potential physiological roles

Pannexins, large non-gap junction super family exists in vertebrates, play multiple roles in different cellular functions through their ATP release. Panx1-mediated adenosine 5′-triphosphate (ATP) release plays a vital role in physiological and pathophysiological conditions and is known major extracellular molecule in purinergic signaling. To modulate their function in vivo, a proper regulation of channel is necessary. Post-translational modifications are considered to be some regulating mechanisms for PANX1, while PANX2, PANX3 have been uncharacterized to date. Through their significant evidences, PANXs exclude from gap junction and conduits ATP release and other cellular molecules from cells by various mechanisms. PANX1 is most extensive characterized and implicated in ATP signaling and inflammatory processes. Despite the constant advances, much significance of PANX1 in physiological processes remains elusive. Recently, various research groups along with our group have reported the Cryo-EM structure of Panx1 channel and uncovered the hidden functions in structure–function mechanism as well as to provide the clear understanding in physiological and pathophysiological roles. These research groups reported the novel heptameric structure with contains 4 transmembrane helices (TM), two extracellular loops and one intracellular loop with N and C terminus located at the intracellular side. In addition, the structure contains a large pore of which an inhibitor CBX act as a plug that blocking the passage of substrate. In this context, this review will present current mechanistic understanding in structure and function together with significant physiological roles particularly ATP release in health and disease. As such, this review emphasizes on recent functional properties associated with novel heptameric channel and demystifies channel-mediated ATP release function.

     

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.     

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.     

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.     

Pore positioning: Current concepts in Pannexin channel trafficking

Pannexins (Panxs) are a multifaceted family of ion and metabolite channels that play key roles in a number of physiological and pathophysiological settings. These single membrane large-pore channels exhibit a variety of tissue, cell type, and subcellular distributions. The lifecycles of Panxs are complex, yet must be understood to accurately target these proteins for future therapeutic use. Here we review the basics of Panx function and localization, and then analyze the recent advances in knowledge regarding Panx trafficking. We examine several intrinsic features of Panxs including specific post-translational modifications, the divergent C-termini, and oligomerization, all of which contribute to Panx anterograde transport pathways. Further, we examine the potential influence of extrinsic factors, such as protein-protein interactions, on Panx trafficking. Finally, we highlight what is currently known with respect to Panx internalization and retrograde transport, and present new data illustrating Panx1 internalization following an activating stimulus.     

Connexins and pannexins in neuronal development and adult neurogenesis

Connexins and pannexins share very similar structures and functions; they also exhibit overlapping expression in many stages of neuronal development. Here, we review evidence implicating connexin- and pannexin-mediated communication in the regulation of the birth and development of neurons, specifically Cx26, Cx30, Cx32, Cx36, Cx43, Cx45, Panx1, and Panx2. We begin by dissecting the involvement of these proteins in the generation and development of new neurons in the embryonic, postnatal, and adult brain. Next we briefly outline common mechanisms employed by both pannexins and connexins in these roles, including modulation of purinergic receptor signalling and signalling nexus functions. Throughout this review we highlight developing themes as well as important gaps in knowledge to be bridged.

     

Pannexin channels in ATP release and beyond: An unexpected rendezvous at the endoplasmic reticulum

The pannexin (Panx) family of proteins, which is co-expressed with connexins (Cxs) in vertebrates, was found to be a new GJ-forming protein family related to invertebrate innexins. During the past ten years, different studies showed that Panxs mainly form hemichannels in the plasma membrane and mediate paracrine signalling by providing a flux pathway for ions such as Ca²⁺, for ATP and perhaps for other compounds, in response to physiological and pathological stimuli. Although the physiological role of Panxs as a hemichannel was questioned, there is increasing evidence that Panx play a role in vasodilatation, initiation of inflammatory responses, ischemic death of neurons, epilepsy and in tumor suppression. Moreover, it is intriguing that Panxs may also function at the endoplasmic reticulum (ER) as intracellular Ca²⁺-leak channel and may be involved in ER-related functions. Although the physiological significance and meaning of such Panx-regulated intracellular Ca²⁺ leak requires further exploration, this functional property places Panx at the centre of many physiological and pathophysiological processes, given the fundamental role of intracellular Ca²⁺ homeostasis and dynamics in a plethora of physiological processes. In this review, we therefore want to focus on Panx as channels at the plasma membrane and at the ER membranes with a particular emphasis on the potential implications of the latter in intracellular Ca²⁺ signalling.     

Connexins and pannexins in neuronal development and adult neurogenesis

Connexins and pannexins share very similar structures and functions; they also exhibit overlapping expression in many stages of neuronal development. Here, we review evidence implicating connexin- and pannexin-mediated communication in the regulation of the birth and development of neurons, specifically Cx26, Cx30, Cx32, Cx36, Cx43, Cx45, Panx1, and Panx2. We begin by dissecting the involvement of these proteins in the generation and development of new neurons in the embryonic, postnatal, and adult brain. Next we briefly outline common mechanisms employed by both pannexins and connexins in these roles, including modulation of purinergic receptor signalling and signalling nexus functions. Throughout this review we highlight developing themes as well as important gaps in knowledge to be bridged.     

Pore positioning

Pannexins (Panxs) are a multifaceted family of ion and metabolite channels that play key roles in a number of physiological and pathophysiological settings. These single membrane large-pore channels exhibit a variety of tissue, cell type, and subcellular distributions. The lifecycles of Panxs are complex, yet must be understood to accurately target these proteins for future therapeutic use. Here we review the basics of Panx function and localization, and then analyze the recent advances in knowledge regarding Panx trafficking. We examine several intrinsic features of Panxs including specific post-translational modifications, the divergent C-termini, and oligomerization, all of which contribute to Panx anterograde transport pathways. Further, we examine the potential influence of extrinsic factors, such as protein-protein interactions, on Panx trafficking. Finally, we highlight what is currently known with respect to Panx internalization and retrograde transport, and present new data illustrating Panx1 internalization following an activating stimulus.     

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.     

Connexin Channels at the Glio-Vascular Interface: Gatekeepers of the Brain

Neuronal survival, electrical signaling and synaptic activity require a well-balanced micro-environment in the central nervous system. This is achieved by the blood–brain barrier (BBB), an endothelial barrier situated in the brain capillaries, that controls near-to-all passage in and out of the brain. The endothelial barrier function is highly dependent on signaling interactions with surrounding glial, neuronal and vascular cells, together forming the neuro-glio-vascular unit. Within this functional unit, connexin (Cx) channels are of utmost importance for intercellular communication between the different cellular compartments. Connexins are best known as the building blocks of gap junction (GJ) channels that enable direct cell–cell transfer of metabolic, biochemical and electric signals. In addition, beyond their role in direct intercellular communication, Cxs also form unapposed, non-junctional hemichannels in the plasma membrane that allow the passage of several paracrine messengers, complementing direct GJ communication. Within the NGVU, Cxs are expressed in vascular endothelial cells, including those that form the BBB, and are eminent in astrocytes, especially at their endfoot processes that wrap around cerebral vessels. However, despite the density of Cx channels at this so-called gliovascular interface, it remains unclear as to how Cx-based signaling between astrocytes and BBB endothelial cells may converge control over BBB permeability in health and disease. In this review we describe available evidence that supports a role for astroglial as well as endothelial Cxs in the regulation of BBB permeability during development as well as in disease states.     

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.     

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.     

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.     

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.     

Pannexin 3 functions as an ER Ca(2+) channel, hemichannel, and gap junction to promote osteoblast differentiation

The pannexin proteins represent a new gap junction family. However, the cellular functions of pannexins remain largely unknown. Here, we demonstrate that pannexin 3 (Panx3) promotes differentiation of osteoblasts and ex vivo growth of metatarsals. Panx3 expression was induced during osteogenic differentiation of C2C12 cells and primary calvarial cells, and suppression of this endogenous expression inhibited differentiation. Panx3 functioned as a unique Ca(2+) channel in the endoplasmic reticulum (ER), which was activated by purinergic receptor/phosphoinositide 3-kinase (PI3K)/Akt signaling, followed by activation of calmodulin signaling for differentiation. Panx3 also formed hemichannels that allowed release of ATP into the extracellular space and activation of purinergic receptors with the subsequent activation of PI3K-Akt signaling. Panx3 also formed gap junctions and propagated Ca(2+) waves between cells. Blocking the Panx3 Ca(2+) channel and gap junction activities inhibited osteoblast differentiation. Thus, Panx3 appears to be a new regulator that promotes osteoblast differentiation by functioning as an ER Ca(2+) channel and a hemichannel, and by forming gap junctions.