The P2X7 receptor–pannexin connection to dye uptake and IL-1β release
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- Pelegrin, P. & Surprenant, A. Purinergic Signalling (2009) 5: 129. doi:10.1007/s11302-009-9141-7
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The P2X7 receptor (P2X7R) is uniquely associated with two distinct cellular responses: activation of a dye-permeable pathway allowing passage of molecules up to 900 Da and rapid release of the pro-inflammatory cytokine, interleukin-1β (IL-1β), from activated macrophage. How this dye uptake path forms and whether it is involved in IL-1β release has not been known. Pannexin-1 is a recently identified protein found to physically associate with the P2X7R. Inhibition of pannexin-1 does not alter P2X7R ion channel activation or associated calcium flux but blocks one component of P2X7R-induced dye uptake and unmasks a slower, previously undetected, dye uptake pathway. Inhibition of pannexin-1 blocks P2X7R-mediated IL-1β release from macrophage as well as release mediated by other stimuli which couple to activation of capase-1 and additionally inhibits the release of interleukin-1α, a member of the IL-1 family whose processing does not require caspase-1 activation. Thus, pannexin-1 is linked to both dye uptake and IL-1β release but via distinct mechanisms.
The first report of extracellular adenosine triphosphate (ATP) as a “cell-permeabilizing” agent was in 1975 when Rozengurt and Heppel  found that p-nitrophenyl phosphate entered transformed 3T3 cells during a 5-min exposure to ATP. Then, in 1979 Cockcroft and Gomperts  similarly found that inorganic phosphates rapidly entered mast cells upon ATP stimulation. Through the 1980s, work by the groups of Silverstein [3–7], Weisman [8–11], and Wiley [12–14] provided pharmacological, biophysical, and biochemical characterization of what became known as the P2Z receptor , which was particularly prominent in immune cells. The most striking cellular feature that clearly separated P2Z receptor activation from other purine receptors was the rapid uptake of higher molecular weight molecules (up to ∼900 Da). This cell permeabilization process was considered to be due to the opening of a non-selective “large pore” in contrast to the cationic channels that formed the P2X receptor subtype . The P2Z receptor in macrophage and lymphocytes became a potential anti-inflammatory drug target by the early 1990s with studies by the groups of Chaplin , Gabel [17–19], Di Virgilio [20–24], and Dubyak [12, 25–29] demonstrating that ATP, most likely acting on P2Z receptors, was the most potent physiological stimulus for the rapid release of the pro-inflammatory cytokine, interleukin-1β (IL-1β), from activated monocytes and macrophages. Soon after the Glaxo-Geneva group discovered the molecular identity of the P2Z receptor as the P2X7 receptor (P2X7R) in 1996/1997 [30, 31], high throughput screening on heterologously expressed, or endogenous, P2X7 receptors using dye uptake assays was begun by virtually all Big Pharma companies [32, 33]. During this first decade of the twenty-first century, several highly selective and potent P2X7R antagonists have been discovered and found to be effective in animal models of neuropathic pain and other inflammatory processes, most likely through a reduced release of bioactive IL-1β and other members of the IL-1 family of pro-inflammatory cytokines (IL-1α and IL-18) . Most recently, AstraZeneca have reported initial positive results from phase II clinical trials of their P2X7R antagonist in the treatment of rheumatoid arthritis . Thus, the practical link between the P2X7R-mediated “dye uptake/large pore” path and IL-1β release inflammatory processes in regard to drug discovery is clear. But is there a physiological link, and, if there is, what is the underlying mechanism by which the dye uptake path signals to IL-1 processing and release? This is the main question we will address in this review. We will focus on results obtained from studies of endogenous P2X7R in monocytes and macrophage and of heterologously expressed P2X7R in mammalian cells because few, if any, differences have been found when comparing data obtained from these systems. Although P2X7R functions in other immune cells (neutrophils, B and T lymphocytes) are generally similar to macrophage, there are some distinct differences that may suggest alternative signaling pathways and/or interacting protein complexes; these will not be considered here.
P2X7R ion channel and large pore
Evidence for P2X7R ion channel itself as the large pore
Evidence against P2X7R ion channel itself as the large pore
Certain observations were inconsistent with the hypothesis that the P2X7R ion channel dilates to allow passage of larger molecules. Firstly, very low concentrations of the calmodulin inhibitor calmidazolium were shown to inhibit the P2X7R ion channel by up to 95% yet did not decrease dye uptake . Secondly, it was not clear how anionic dyes, such as Lucifer yellow (MW 457), which are well established as being taken up by cells in response to P2X7R activation [3, 6, 37, 38], could permeate the cation-selective channel. Thirdly, specific deletions or mutations in the C-terminal domain of the P2X7R were made that completely blocked pore dilatation as measured by NMDG+ permeability shifts (Fig. 1c, d) yet dye uptake and membrane currents were both significantly enhanced  (Fig. 1e). Moreover, although significant YO-PRO-1 uptake was observed in normal extracellular sodium concentrations, there was no NMDG permeability increase observed, thus dissociating NMDG permeability changes from dye uptake . Finally, low micromolar concentrations of the gap junction blocker carbenoxolone (CBX) markedly inhibited P2X7R dye uptake without altering membrane currents or initial calcium flux . These results suggested two other possibilities: that P2X7R channel activation induces a distinct signal transduction pathway which leads to dye uptake , or a distinct P2X7R-interacting protein is the dye uptake pathway .
Pannexin-1 mediates rapid dye uptake pathway
Pannexins were originally identified in 2003/2004 by low sequence homology to invertebrate gap junction channels, the innexins [44-46]. Like innexins and the mammalian gap junction channels, connexins, the three members of the pannexin family (panx1, 2, 3) share a similar membrane topology consisting of four transmembrane domains, short extracellular segments, and intracellular C and N termini [44–46]. Pannexins do not show sequence homology to the large family of connexin proteins, although recent detailed phylogenetic analysis has convincingly placed innexins, pannexins, and connexins within the same molecular superfamily . In situ hybridization and immunohistochemistry at both light and electron microscopy levels show that panx1 is widely expressed, particularly in immune cells, endothelia, and epithelia; panx2 is relatively neuronal-specific, while panx3 shows fairly localized expression to joints and skin [45, 46, 48]. Gap junctions are composed of 12 connexin proteins via hexameric connexin complexes (called connexons or hemichannels) on two adjacent cells coming together to form a junctional channel through which ions and small molecular weight (up to 1,000 Da) molecules can pass . Initial studies in which panx1 was over-expressed in oocytes suggested panx1 could act to form gap junctions in a manner similar to that known for connexins [45, 48], but no gap junction formation has been observed when panx1 is expressed in mammalian cells [40, 49] and the current understanding is that panx1 does not form gap junctions [49, 50].
When panx1 is ectopically expressed in HEK 293 cells lacking P2X7R, a low level of constitutive dye uptake occurs which is not otherwise observed in untransfected or vector-transfected cells nor in P2X7R-expressing cells in the absence of ATP . We have also noted a significant level of constitutive dye uptake in peritoneal macrophage obtained from P2X7R−/− mice that is not seen in wild-type macrophage (unpublished observations). These findings may suggest that panx1 is normally under negative control when it is in association with unstimulated P2X7R.
How panx1 mediates dye uptake has not been resolved although it is currently assumed that panx1 forms a large conductance hemichannel in the plasma membrane through which cationic and anionic molecules of up to 800–900 Da can pass [46, 49, 50]. This hypothesis is largely based on analogy to gap junction channels rather than to direct experimental evidence. The main evidence that panx1 acts as a large conductance hemichannel is that when panx1 is heterologously expressed (in the absence of P2X7R), a cation/anion non-selective membrane current can be recorded [40, 45]. However, there are critical questions that must be resolved before panx1 can be considered a plasma membrane hemichannel through which marker dyes directly pass. (1) Single channel recordings showing large conductance unitary opening are critically required. We have consistently failed to record large conductance single channels from panx1-expressing, or P2X7R/panx1 over-expressed, mammalian cells; there is one report of large conductance single channels (>200 pS) being observed in oocytes over-expressing panx1 , but this does not appear to be a consistent observation. (2) Site-directed mutagenesis of residues within the panx1 protein that result in altered voltage dependence, ion permeability, and/or unitary conductance are required to provide direct demonstration that panx1 is, itself, an ion channel. (3) If a large conductance pannexin channel is activated by P2X7R stimulation, then one would expect considerable current inhibition in the ATP-induced current in response to P2X7R stimulation. But, although CBX and 10panx1 inhibitory peptide completely block the membrane current recorded when panx1 is ectopically expressed (in the absence of P2X7R), they have no effect or slightly enhance the membrane current activated by P2X7R stimulation in cells expressing both panx1 and P2X7R . (4) It remains unclear whether endogenous panx1 is a plasma membrane protein or an endoplasmic reticulum (ER) membrane protein or both. There is evidence that panx1 may be an ER membrane protein involved in ER calcium regulation . There are currently no studies showing high-resolution subcellular localization of endogenous panx1 in macrophage using antibodies that are highly specific for panx1. (5) Can endogenous panx1-like currents be recorded from macrophage? There are no reports of membrane currents recorded from monocytes or macrophage (or any neuronal or non-neuronal cell) having properties similar to those observed when panx1 is over-expressed in HEK 293 or other mammalian cells. Thus, until these questions are answered, alternative possibilities must be considered, in particular the possibility that panx1 may recruit, or activate, transporter proteins which provide the direct route for entry and exit of dyes and other small molecules.
P2X7R–pannexin-1 and IL-1β processing and release
How might panx1 signal to activate the NLRP3 inflammasome in response to P2X7R stimulation, or to dye uptake independent stimuli? The classic hypothesis for activation of caspase-1 via the NLRP3 inflammasome is the K+ efflux model . This model is based on three main observations: (a) activation of P2X7R in macrophages induces K+ depletion [17, 40, 53, 57] as does stimulation with nigericin or maitotoxin; (b) activation of caspase-1 by these stimuli does not occur in high extracellular K+ solution [40, 57–59]; and (c) the in vitro assembly of the inflammasome is dependent on K+ concentrations lower than 70 mM . Initially, panx1 was expected to be a conduit for the high K+ efflux subsequent to P2X7R activation, but inhibition of panx1 using 10panx1 inhibitory peptide abolished caspase-1 activation without altering P2X7R-mediated K+ efflux . This result implies that panx1 signals downstream to K+ efflux and that K+ efflux is independent of panx1-mediated macrophage permeabilization and dye uptake. It is also possible that the panx1 inhibitory peptide may block downstream panx1 signaling without altering direct K+ permeability through panx1 channels. However, panx1 inhibition does not alter membrane currents or calcium influx in response to P2X7R activation (see above). Taken together, it seems most likely that K+ efflux occurs through the P2X7R ion channel itself and that panx1 acts on the inflammasome downstream of ion flux. This idea that panx1 is unlikely to be directly involved with ion flux in macrophage is further supported by a recent study where inhibition of panx1 by CBX prevented ATP-mediated IL-1β release from macrophage, as expected, but did not alter concurrent release of cathepsin B, this latter process resulting from a Ca++-dependent exocytosis of secretory lysosomes .
If K+ efflux is not the link between panx1 and inflammasome activation, an alternative hypothesis comes from the group of Nuñez who have suggested that panx1 activation by P2X7R may act to deliver PAMPs directly into the cytosol where they can then directly bind to leucine-rich repeats present in Nod-like receptors (i.e., NLRP3) to directly activate the inflammasome [61, 62]. This is an attractive idea whereby PAMPs would be not only responsible for the initial inflammatory stimulus by activating the TLR–NFκB synthetic pathway but also for the secondary stimulus leading to processing and release of IL-1β via panx1-mediated delivery into the cell interior. This novel hypothesis is based on observations that cytosolic delivery of the bacterial PAMP lysosomic muramyl dipeptide (MDP) activated the inflammasome in a manner similar to P2X7R and that P2X7R activation induced a panx1-dependent lysosome-to-cytosol translocation of MDP [61, 62]. However, there are three major difficulties in this hypothesis as a generalized mechanism for inflammasome activation. Firstly, it is not clear how a P2X7R/panx1 permeabilization pore that is presumably limited to molecules <900 Da can transfer larger PAMPs. That is, while PAMPs like MDP (MW 492 Da) may be expected to pass, other PAMPs are too large, particularly the classically used lipopolysaccharide (LPS) whose active fragment, lipid A, has a molecular mass of 1,700–1,800 Da (depending on the number and identity of fatty acid chains present) . Secondly, most IL-1 cytokine release experiments are performed by initial incubation with LPS or other PAMPs followed by washing away these PAMPs and applying ATP to activate P2X7R [40, 58, 60]. Therefore, minimal levels of PAMPs are likely to be present in the ATP incubation media for delivery into the cell. Thirdly, the studies using different PAMPs to activate the inflammasome [61, 62] have been carried out in the presence of ATP, thus release of bioactive IL-1β release must be in part—or in toto—due to P2X7R activation.
A third hypothesis regarding panx1-mediated activation of the inflammasome is that extracellular ATP activates the P2X7R, which in turn activates panx1 hemichannel (or transporter) activity to directly transport ATP into the cell. Evidence has been presented to suggest panx1 can act as a conduit for ATP release from erythrocytes . If panx1 is a conduit for ATP, it must be a conduit for either entry or exit of ATP depending on the concentration gradient. Concentrations of ATP required to activate P2X7R (usually >100 μM) may be higher than free intracellular ATP in localized regions near the plasma membrane, especially as the vast majority of intracellular ATP (∼90%) is produced in, and bound to, mitochondria . Importantly, ATP can directly activate capase-1 in cell-free systems , and elevations of intracellular ATP levels have been associated with, and required for, caspase-1 activation and IL-1β release [67, 68]. This is an attractive model for P2X7R-mediated inflammasome activation although there is currently no direct or indirect experimental evidence for or against this hypothesis. However, this hypothesis also cannot be a generalized mechanism for panx1 involvement in caspase-1-dependent IL-1β processing and release because other stimuli, such as nigericin and maitotoxin, that are equally panx1-dependent, do not require extracellular ATP.
Recent work in our lab suggests panx1 may have a wider role in pro-inflammatory cytokine release beyond initial involvement in caspase-1 activation. In an investigation of the kinetics of action of 10panx1-mimetic inhibitory peptide, we found that this peptide was able to stop further IL-1β release from macrophage after caspase-1/inflammasome activation had already been instigated by P2X7R stimulation. The 10panx1 peptide did not inhibit caspase-1 activity per se in cell-free assays, so ruling out a direct chemical or enzymatic interaction with caspase-1. Moreover, inhibition of panx1 not only inhibited caspase-1-dependent processing and release of both IL-1β and IL-18 but also IL-1α which does not directly require caspase-1 inflammasome activation for its processing . Additionally, a significant practical finding was that inhibition lasted for >1 h after removal of the inhibitory peptide, thus indicating tight, long-lasting binding and/or prolonged inhibition of unknown processes. In this regard, it is important to note that prolonged incubation (>2–3 h) with the 10panx1 inhibitory peptide is toxic to macrophage . Although the mechanism of 10panx1-mimetic toxicity during prolonged application has not been investigated, it clearly differs from the rapid (non-toxic) inhibition of caspase-1 activation that occurs with brief (<30 min) applications of this inhibitor [40, 42, 56]. These results make it clear that this compound should be used with caution, and investigators should ensure that appropriate optimization protocols (concentration and time dependence) are carried out when employing this inhibitor. This is particularly true in view of the well-known variability in potency/purity of commercially synthesized peptides and proteins.
P2X7R activation leads to numerous downstream signaling events: the most upstream event subsequent to initial ion channel opening is induction of the large pore/dye uptake path that occurs approximately 2–3 s after receptor activation . The most physiologically relevant downstream event is the release of the pro-inflammatory group of IL-1 cytokines, IL-1α, IL-1β, and IL-18; cytokine release can be measured by ELISA or Western blot techniques within 5–15 min of receptor activation [60, 69, 70] although it is likely that release occurs even sooner. Recent studies summarized in this review have identified panx1 as a critical component in both these events, but it appears that the panx1-dependent dye uptake path is not the causative event underlying cytokine release. Is this dye uptake event that has been so extensively utilized since the original studies in the mid-1970s simply a functionally meaningless by-product of P2X7R–panx1 activation? If so, it has been an enormously practical by-product by providing a highly sensitive and selective screening assay that has resulted in the identification of potent P2X7R antagonists that are now of promising therapeutic value in the treatment of inflammatory diseases [32–34]. Or, if the dye uptake event does represent a functionally relevant mechanism underlying P2X7R function, we have still to discover what this is. Speculative hypotheses have been discussed in this review and are depicted in Fig. 3. It is likely that some, or all, of these hypotheses will be revised, discarded, or perhaps even shown to be valid, but certainly, they provide exciting impetus for much further exploration of the P2X7R–pannexin connection.
Work in our lab is funded by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council of Britain, and AstraZeneca Charnwood.
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