Arachidonic acid closes innexin/pannexin channels and thereby inhibits microglia cell movement to a nerve injury

Pannexons are membrane channels formed by pannexins and are permeable to ATP. They have been implicated in various physiological and pathophysiological processes. Innexins, the invertebrate homologues of the pannexins, form innexons. Nerve injury induces calcium waves in glial cells, releasing ATP through glial pannexon/innexon channels. The ATP then activates microglia. More slowly, injury releases arachidonic acid (ArA). The present experiments show that ArA itself reduced the macroscopic membrane currents of innexin‐ and of pannexin‐injected oocytes; ArA also blocked K⁺‐induced release of ATP. In leeches, whose large glial cells have been favorable for studying control of microglia migration, ArA blocked glial dye‐release and, evidently, ATP‐release. A physiological consequence in the leech was block of microglial migration to nerve injuries. Exogenous ATP (100 µM) reversed the effect, for ATP causes activation and movement of microglia after nerve injury, but nitric oxide directs microglia to the lesion. It was not excluded that metabolites of ArA may also inhibit the channels. But for all these effects, ArA and its non‐metabolizable analog eicosatetraynoic acid (ETYA) were indistinguishable. Therefore, ArA itself is an endogenous regulator of pannexons and innexons. ArA thus blocks release of ATP from glia after nerve injury and thereby, at least in leeches, stops microglia at lesions. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 621–631, 2013     

Connexin and pannexin channels in cancer

Communication among cells via direct cell-cell contact by connexin gap junctions, or between cell and extracellular environment via pannexin channels or connexin hemichannels, is a key factor in cell function and tissue homeostasis. Upon malignant transformation in different cancer types, the dysregulation of these connexin and pannexin channels and their effect in cellular communication, can either enhance or suppress tumorigenesis and metastasis. In this review, we will highlight the latest reports on the role of the well characterized connexin family and its ability to form gap junctions and hemichannels in cancer. We will also introduce the more recently discovered family of pannexin channels and our current knowledge about their involvement in cancer progression.     

Connexin and pannexin channels in cancer

Communication among cells via direct cell-cell contact by connexin gap junctions, or between cell and extracellular environment via pannexin channels or connexin hemichannels, is a key factor in cell function and tissue homeostasis. Upon malignant transformation in different cancer types, the dysregulation of these connexin and pannexin channels and their effect in cellular communication, can either enhance or suppress tumorigenesis and metastasis. In this review, we will highlight the latest reports on the role of the well characterized connexin family and its ability to form gap junctions and hemichannels in cancer. We will also introduce the more recently discovered family of pannexin channels and our current knowledge about their involvement in cancer progression.

     

Innexin and pannexin channels and their signaling

Innexins are bifunctional membrane proteins in invertebrates, forming gap junctions as well as non-junctional membrane channels (innexons). Their vertebrate analogues, the pannexins, have not only lost the ability to form gap junctions but are also prevented from it by glycosylation. Pannexins appear to form only non-junctional membrane channels (pannexons). The membrane channels formed by pannexins and innexins are similar in their biophysical and pharmacological properties. Innexons and pannexons are permeable to ATP, are present in glial cells, and are involved in activation of microglia by calcium waves in glia. Directional movement and accumulation of microglia following nerve injury, which has been studied in the leech which has unusually large glial cells, involves at least 3 signals: ATP is the “go” signal, NO is the “where” signal and arachidonic acid is a “stop” signal.     

Regulation of pannexin channels by post-translational modifications

The large-pore channels formed by the pannexin family of proteins have been implicated in many physiological and pathophysiological functions, mainly through their ATP release function. However, a tight regulation of channel opening is necessary to modulate their function in vivo. Post-translational modifications have been postulated as some of the regulating mechanisms for Panx1, while Panx2 and Panx3 have not been as well characterized. Positive regulators include caspase cleavage to open Panx1 channels in apoptotic cells, and activation by Src family kinases via ionotropic receptors in neurons and macrophages. S-nitrosylation of cysteines has been shown to both inhibit and activate the Panx1 channel in different cell types. All three pannexins are N-glycosylated but to different levels of modification. Their diverse glycosylation appears to regulate cellular localization, intermixing, and may restrict their ability to function as inter-cellular channels. It is clear that our understanding of pannexin post-translational modification and their role in channel function regulation is still in its infancy even a decade after their discovery.     

Diverse subcellular distribution profiles of pannexin 1 and pannexin 3

Pannexins have been proposed to play a role in gap junctional intercellular communication and as single-membrane channels, although many of their molecular characteristics differ from connexins. Localization of untagged Panx1 and Panx3 exogenously expressed in five cultured cell lines revealed a cell surface distribution profile with limited evidence of cell surface clustering and variable levels of intracellular pools. However, N-glycosylation-defective mutants of pannexins exhibited a more prominent intracellular distribution with decreased cell surface labeling, suggesting an important role for pannexin glycosylation in trafficking. Similar to wild-type pannexins, the glycosylation-defective mutants failed to noticeably transfer microinjected fluorescent dyes to neighboring cells, suggesting that few, or no functional intercellular channels were formed. Finally, varied distribution patterns of endogenous Panx1 and Panx3 were observed in cells of osteoblast origin and Madin-Darby canine kidney cells. Collectively, diverse expression and distribution profiles of Panx1 and Panx3 suggest that they may have multiple cellular functions.     

A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP

Pannexin 1 forms a large membrane channel that, based on its biophysical properties and its expression pattern, is a prime candidate to represent an ATP release channel. Pannexin 1 channel activity is potentially deleterious for cells as indicated by its involvement in the P2X7 death complex. Here we describe a negative feedback loop controlling pannexin 1 channel activity. ATP, permeant to pannexin 1 channels, was found to inhibit its permeation pathway when applied extracellularly to oocytes expressing pannexin 1 exogenously. ATP analogues, including benzoylbenzoyl-ATP, suramin, and brilliant blue G were even more effective inhibitors of pannexin 1 currents than ATP. These compounds also attenuated the uptake of dyes by erythrocytes, which express pannexin 1. The rank order of the compounds in attenuation of pannexin 1 currents was similar to their binding affinities to the P2X7 receptor, except that receptor agonists and antagonists both were inhibitory to the channel. Mutational analysis identified R75 in pannexin 1 to be critical for ATP inhibition of pannexin 1 currents.     

Differentiating connexin hemichannels and pannexin channels in cellular ATP release

Adenosine triphosphate (ATP) plays a fundamental role in cellular communication, with its extracellular accumulation triggering purinergic signaling cascades in a diversity of cell types. While the roles for purinergic signaling in health and disease have been well established, identification and differentiation of the specific mechanisms controlling cellular ATP release is less well understood. Multiple mechanisms have been proposed to regulate ATP release with connexin (Cx) hemichannels and pannexin (Panx) channels receiving major focus. However, segregating the specific roles of Panxs and Cxs in ATP release in a plethora of physiological and pathological contexts has remained enigmatic. This multifaceted problem has arisen from the selectivity of pharmacological inhibitors for Panxs and Cxs, methodological differences in assessing Panx and Cx function and the potential compensation by other isoforms in gene silencing and genetic knockout models. Consequently, there remains a void in the current understanding of specific contributions of Panxs and Cxs in releasing ATP during homeostasis and disease. Differentiating the distinct signaling pathways that regulate these two channels will advance our current knowledge of cellular communication and aid in the development of novel rationally-designed drugs for modulation of Panx and Cx activity, respectively.     

Manoalide, a natural sesterterpenoid that inhibits calcium channels

Manoalide is a marine natural product that has anti-inflammatory and anti-proliferative activities and is an irreversible inhibitor of phospholipase A2 and phospholipase C. It is now shown that the compound is a potent inhibitor of Ca2+ mobilization in several cell types. In A431 cells the increase in epidermal growth factor receptor-mediated Ca2+ entry and release from intracellular Ca2+ stores were blocked by manoalide in a time-dependent manner with an IC50 of 0.4 microM. The effect of manoalide on phosphoinositide metabolism, namely the production of inositol monophosphate, did not coincide with its effect on the epidermal growth factor response. In GH# cells, manoalide blocked the thyrotropin-releasing hormone-dependent release of Ca2+ from intracellular stores without inhibition of the formation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate. Manoalide also blocked the K+ depolarization-activated Ca2+ channel in these cells as well as the activation of the channel by Bay K8644 with an IC50 of 1 microM. In addition, manoalide also inhibited the Ca2+ influx induced by concanavalin A in mouse spleen cells in a time- and temperature-sensitive manner with an IC50 of 0.07 microM. However, neither forskolin-activated adenylate cyclase in A431 cells nor the distribution of the potential sensitive dye, 3,3'-dipropylthiodicarbocyanide iodide in GH3 cells was affected by manoalide. Thus, manoalide acts as a Ca2+ channel inhibitor in all cells examined. This action may account for its effects on inflammation and proliferation and may be independent of its effect on phospholipases.     

Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium

The ability for long-range communication through intercellular calcium waves is inherent to cells of many tissues. A dual propagation mode for these waves includes passage of IP3 through gap junctions as well as an extracellular pathway involving ATP. The wave can be regenerative and include ATP-induced ATP release via an unknown mechanism. Here, we show that pannexin 1 channels can be activated by extracellular ATP acting through purinergic receptors of the P2Y group as well as by cytoplasmic calcium. Based on its properties, including ATP permeability, pannexin 1 may be involved in both initiation and propagation of calcium waves.     

Targeting pannexin1 improves seizure outcome

Imbalance of the excitatory neurotransmitter glutamate and of the inhibitory neurotransmitter GABA is one of several causes of seizures. ATP has also been implicated in epilepsy. However, little is known about the mechanisms involved in the release of ATP from cells and the consequences of the altered ATP signaling during seizures. Pannexin1 (Panx1) is found in astrocytes and in neurons at high levels in the embryonic and young postnatal brain, declining in adulthood. Panx1 forms large-conductance voltage sensitive plasma membrane channels permeable to ATP that are also activated by elevated extracellular K(+) and following P2 receptor stimulation. Based on these properties, we hypothesized that Panx1 channels may contribute to seizures by increasing the levels of extracellular ATP. Using pharmacological tools and two transgenic mice deficient for Panx1 we show here that interference with Panx1 ameliorates the outcome and shortens the duration of kainic acid-induced status epilepticus. These data thus indicate that the activation of Panx1 in juvenile mouse hippocampi contributes to neuronal hyperactivity in seizures.     

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.     

Analysis of a pannexin 2-pannexin 1 chimeric protein supports divergent roles for pannexin C-termini in cellular localization

Abstract

Pannexins (Panxs) are a three-member family of large pore ion channels permeable to ions and small molecules. Recent elegant work has demonstrated that the Panx1 C-terminus plays an important role in channel trafficking. Panx2, another family member, has a longer and highly dissimilar C-terminus. Interestingly, Panx1 is readily found at the plasma membrane, while Panx2 is mainly present on intracellular membranes. Here we used overlap-extension cloning to create the first chimeric Panx, consisting of Panx2 with the Panx1 C-terminus (Panx2^Panx1CT), to determine whether the Panx1 C-terminus influences the trafficking of Panx2. We are the first to observe a high level of co-localization between Panx2 and the endolysosomal enriched mannose-6-phosphate receptor. Interestingly this distinct localization of Panx2 is altered by the presence of the Panx1 C-terminus. These novel observations support previous data indicating the importance of the C-terminus in the control of Panx trafficking, and highlight the complexity of molecular signals involved.     

Trafficking dynamics of glycosylated pannexin 1 proteins

Pannexins are mammalian orthologs of innexins and have a predicted topological folding pattern similar to that of connexins, except they are glycosylated. Rat pannexin 1 is glycosylated at N254 and this residue is important for plasma membrane targeting. Here we demonstrate that cell surface expression levels of the rat pannexin 1 N254Q mutant are rescued by coexpression with the wild-type protein. In paired Xenopus oocytes, the functional effect of this rescue is inconsequential; however, cell surface deglycosylation by PNGase F significantly enhanced functional gap junction formation. In mammalian cells, wild-type oligomers traffic at a slower rate than Myc-or tetracysteine domain-tagged versions, a behavior opposite to that of tagged connexins. The temporal differences of Panx1 trafficking correlate with spatial differences of intracellular localizations induced by Golgi blockage by Brefeldin-A or glycosylation prevention by tunicamycin. Therefore, Panx1 has kinetics and dynamics that make it unique to serve distinct functions separate from connexin-based channels.     

Identification of a potential regulator of the gap junction protein pannexin1

Recent studies have revealed a second class of gap-junction-forming proteins in vertebrates. These genes are termed pannexins, and it has been suggested that they perform similar functions as connexins. Pannexin1 is expressed in diverse tissues including the central nervous system and seems to form gap junction channels in the Xenopus oocyte expression system. Since protein interacting partners have frequently been described for connexins, the most prominent family of gap junction forming proteins, we thus started to search for candidate genes of pannexin interacting partners. Kvbeta3, a protein belonging to the family of regulatory beta-subunits of the voltage-dependent potassium channels, was identified as a binding partner of pannexin1 in an E. coli two-hybrid system. This result was verified by confocal laser scanning microscopy using double transfected Neuro2A cells. The colocalization of both proteins at the plasma membrane is suggestive of functional interaction.     

Mechanisms of ATP release in pain: role of pannexin and connexin channels

Pain is a physiological response to bodily damage and serves as a warning of potential threat. Pain can also transform from an acute response to noxious stimuli to a chronic condition with notable emotional and psychological components that requires treatment. Indeed, the management of chronic pain is currently an important unmet societal need. Several reports have implicated the release of the neurotransmitter adenosine triphosphate (ATP) and subsequent activation of purinergic receptors in distinct pain etiologies. Purinergic receptors are broadly expressed in peripheral neurons and the spinal cord; thus, purinergic signaling in sensory neurons or in spinal circuits may be critical for pain processing. Nevertheless, an outstanding question remains: what are the mechanisms of ATP release that initiate nociceptive signaling? Connexin and pannexin channels are established conduits of ATP release and have been suggested to play important roles in a variety of pathologies, including several models of pain. As such, these large-pore channels represent a new and exciting putative pharmacological target for pain treatment. Herein, we will review the current evidence for a role of connexin and pannexin channels in ATP release during nociceptive signaling, such as neuropathic and inflammatory pain. Collectively, these studies provide compelling evidence for an important role of connexins and pannexins in pain processing.     

Patterns of heterogeneous expression of pannexin 1 and pannexin 2 transcripts in the olfactory epithelium and olfactory bulb

Pannexins form membrane channels that release biological signals to communicate with neighboring cells. Here, we report expression patterns of pannexin 1 (Panx1) and pannexin 2 (Panx2) in the olfactory epithelium and olfactory bulb of adult mice. In situ hybridization revealed that mRNAs for Panx1 and Panx2 were both expressed in the olfactory epithelium and olfactory bulb. Expression of Panx1 and Panx2 was mainly found in cell bodies below the sustentacular cell layer in the olfactory epithelium, indicating that Panx1 and Panx2 are expressed in mature and immature olfactory neurons, and basal cells. Expression of Panx2 was observed in sustentacular cells in a few locations of the olfactory epithelium. In the olfactory bulb, Panx1 and Panx2 were expressed in spatial patterns. Many mitral cells, tufted cells, periglomerular cells and granule cells were Panx1 and Panx2 positive. Mitral cells located at the dorsal and lateral portions of the olfactory bulb showed weak Panx1 expression compared with those in the medial side. However, the opposite was true for the distribution of Panx2 positive mitral cells. There were more Panx2 mRNA positive mitral cells and granule cells compared to those expressing Panx1. Our findings on pannexin expression in the olfactory system of adult mice raise the novel possibility that pannexins play a role in information processing in the olfactory system. Demonstration of expression patterns of pannexins in the olfactory system provides an anatomical basis for future functional studies.     

Mechanisms of pannexin1 channel gating and regulation

Pannexins are a family of integral membrane proteins with distinct post-translational modifications, sub-cellular localization and tissue distribution. Panx1 is the most studied and best-characterized isoform of this gene family. The ubiquitous expression, as well as its function as a major ATP release and nucleotide permeation channel, makes Panx1 a primary candidate for participating in the pathophysiology of CNS disorders. While many investigations revolve around Panx1 functions in health and disease, more recently, details started emerging about mechanisms that control Panx1 channel activity. These advancements in Panx1 biology have revealed that beyond its classical role as an unopposed plasma membrane channel, it participates in alternative pathways involving multiple intracellular compartments, protein complexes and a myriad of extracellular participants. Here, we review recent progress in our understanding of Panx1 at the center of these pathways, highlighting its modulation in a context specific manner. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve     

The role of connexin and pannexin containing channels in the innate and acquired immune response

Connexin (Cx) and pannexin (Panx) containing channels – gap junctions (GJs) and hemichannels (HCs) – are present in virtually all cells and tissues. Currently, the role of these channels under physiological conditions is well defined. However, their role in the immune response and pathological conditions has only recently been explored. Data from several laboratories demonstrates that infectious agents, including HIV, have evolved to take advantage of GJs and HCs to improve viral/bacterial replication, enhance inflammation, and help spread toxicity into neighboring areas. In the current review, we discuss the role of Cx and Panx containing channels in immune activation and the pathogenesis of several infectious diseases. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Mutant LGI1 inhibits seizure-induced trafficking of Kv4.2 potassium channels

Activity-dependent redistribution of ion channels mediates neuronal circuit plasticity and homeostasis, and could provide pro-epileptic or compensatory anti-epileptic responses to a seizure. Thalamocortical neurons transmit sensory information to the cerebral cortex and through reciprocal corticothalamic connections are intensely activated during a seizure. Therefore, we assessed whether a seizure alters ion channel surface expression and consequent neurophysiologic function of thalamocortical neurons. We report a seizure triggers a rapid (<2h) decrease of excitatory postsynaptic current (EPSC)-like current-induced phasic firing associated with increased transient A-type K(+) current. Seizures also rapidly redistributed the A-type K(+) channel subunit Kv4.2 to the neuronal surface implicating a molecular substrate for the increased K(+) current. Glutamate applied in vitro mimicked the effect, suggesting a direct effect of glutamatergic transmission. Importantly, leucine-rich glioma-inactivated-1 (LGI1), a secreted synaptic protein mutated to cause human partial epilepsy, regulated this seizure-induced circuit response. Human epilepsy-associated dominant-negative-truncated mutant LGI1 inhibited the seizure-induced suppression of phasic firing, increase of A-type K(+) current, and recruitment of Kv4.2 surface expression (in vivo and in vitro). The results identify a response of thalamocortical neurons to seizures involving Kv4.2 surface recruitment associated with dampened phasic firing. The results also identify impaired seizure-induced increases of A-type K(+) current as an additional defect produced by the autosomal dominant lateral temporal lobe epilepsy gene mutant that might contribute to the seizure disorder.