The Elusive P2X7 Macropore

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Review

The Elusive P2X7 Macropore Francesco Di Virgilio,1,* Günther Schmalzing,2 and Fritz Markwardt3 ATP, which is released under pathological conditions and is considered a damage-associated molecular pattern (DAMP), activates P2X7 receptors (P2X7Rs), trimeric plasma membrane ion channels selective for small cations. P2X7Rs are partners in NOD-like receptor containing a pyrin (NLRP3) inflammasome activation and promoters of tumor cell growth. P2X7R overstimulation triggers the ATP-dependent opening of a nonselective plasma membrane pore, known as a ‘macropore’, which allows fluxes of large hydrophilic molecules. The pathophysiological functions of P2X7R are thought to be dependent on activation of this conductance pathway, yet its molecular identity is unknown. Recent reports show that P2X7R permeability to organic solutes is an early and intrinsic property of the channel itself. A better understanding of P2X7R-dependent changes in plasma membrane permeability will allow a rationale development of novel anti-inflammatory and anticancer drugs.

Highlights Extracellular ATP causes reversible permeabilization of mammalian cell plasma membranes due to P2X7Rdependent formation of a large conductance pore (the ‘macropore’). ATP is a major constituent of the inflammatory microenvironment and P2X7R has a key role in inflammation and immunity. Most P2X7[362_TD$IF]R-stimulated immune responses depend on the activation of this permeability pathway, but the underlying mechanism is unknown. Recent electrophysiological and cell biological investigations now converge on a unified mechanistic explanation and provide hints as to the biochemical basis for the endogenous modulation of this pathway.

ATP: From the Universal Energy Currency to a Ubiquitous Extracellular Messenger Extracellular ATP is the universal intracellular energy currency as well as [36_TD$IF]an ubiquitous extracellular messenger with a special homeostatic role [1]. ATP is considered the prototypical DAMP (see Glossary) because it is most commonly used to signal tissue injury or distress in complex multicellular organisms (Figure 1) [2]. The extracellular ATP concentration in healthy tissues is very low (a few nmoles/l), while it increases up to several tens or hundreds of mmoles/l at sites of tissue damage, inflammation, or cancer [3]. The ATP-based signaling system benefits from a remarkable plasticity because the vast array of nucleotide receptor (P2R) subtypes is expressed by almost all cells. The P2R family comprises the P2YR and P2XR subfamilies, with eight and seven members, respectively. P2YRs are seven membrane-spanning, G-protein-coupled receptors, while P2XRs are cation-selective channels [4]. P2XRs have been cloned from Dictyostelium, Schistosoma, and algae, indicating an early appearance in evolution [5]. In higher mammals, P2XRs are mainly implicated in the diffuse signaling network based on extracellular ATP, the only known physiological agonist of this subfamily. The ability of extracellular ATP to trigger dramatic changes in plasma membrane permeability has long been known [6,7]. In 1980, a thorough investigation of ATP-induced plasma membrane permeability increases was carried out that led the authors to postulate the existence of a specific plasma membrane receptor for the fully ionized ATP form (‘the ATP4–[361_TD$IF] receptor’) [8]. An ATPactivated receptor with similar properties was later functionally identified in other cell types and named P2Z. In 1996, P2X7R (previously known as P2Z) was cloned, and its ability to trigger plasma membrane permeabilization to large organic cations confirmed [9]. Here, we discuss the roles of P2X7R in promoting plasma membrane permeabilization and in disease.

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It is anticipated that this new knowledge will have great impact on the design of novel anti-inflammatory drugs.

1

Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 2 Department of Pharmacology and Toxicology, University of Aachen, Aachen, Germany 3 Institute for Physiology, Martin Luther University, Halle/Saale, Germany

*Correspondence: [email protected] (F. Di Virgilio).

https://doi.org/10.1016/j.tcb.2018.01.005 © 2018 Elsevier Ltd. All rights reserved.

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The P2X7 Receptor

Glossary

The human P2X7R gene, located on chromosome 12 at 12q24.31, encodes a 595-amino acid protein comprising a bulky extracellular domain and N- and C-terminal residues both on the cytoplasmic side (Figure 2). P2X7 subunits assemble into a functional homotrimeric receptor (P2X7R). Other ion channels, such as TRPV1, ASICs, P2X2R, and P2X4R, but not P2X1R and P2X3R, can also undergo an increase in conductance that allows the influx of high-molecularmass water-soluble solutes [10,11], but the P2X7R is the plasma membrane receptor where this effect has been best described with high reproducibility. The reason for the different permeability properties might be that P2X2R, P2X4R, and P2X7R are slowly or not at all desensitizing, while P2X1R and P2X3R desensitize quickly. [364_TD$IF]The human P2X7R bearing the E496A loss-of-function mutation [365_TD$IF]is able to carry ion currents, but the short open times of this mutant seem to be responsible for the lack of uptake of the organic cation Yo-Pro (F. Markwardt, 2017, unpublished). Conductance ratios of the human P2X7R for small inorganic and medium-size organic cations were measured by Markwardt and co-workers [12]. Reversibility is one of the most striking features of the P2X7R macropore. Removal of ATP within 10–15 min of the addition allows resealing of the plasma membrane and near-perfect recovery of cell functions, despite the profound perturbation of intracellular ion homeostasis caused by P2X7R opening [13]. Findings from different laboratories show that the long COOHterminal tail is needed for activation of the macropore and, therefore, for plasma membrane permeabilization, but not for channel gating [9,14,15]. However, more recent experiments show that this may not always be the case, depending on the experimental conditions, suggesting that lack of the COOH-terminal tail does not fully abolish but simply slows down macropore formation and, thus, fluorescent dye uptake [16].

Alu RNA: short RNA transcribed from Alu elements (short interspersed nucleotide elements) transcribed by RNA polymerase III. Intracellular Alu RNA accumulation has been described in age-related macular degeneration of the retina. Cathelicidin: bactericidal peptide released by human neutrophils. It kills bacteria by perturbing the phospholipid bilayer; eukaryotic cells are resistant to cathelicidin-mediated lysis. Cholesterol rafts: plasma membrane microdomains enriched in cholesterol. They modulate the assembly of receptors and plasma membrane fluidity. Damage-associated molecular patterns (DAMPs): intracellular molecules that are released following cell stress or injury; they are potent inducers of inflammation. Ethidium: fluorescent positively charged DNA-intercalating dye often used to probe the size of the P2X7R pore. Its molecular mass is 394. Fura-2 free acid: the membraneimpermeant acidic form of the wellknown ratiometric fluorescent Ca2+ indicator that, as the acetoxymethyl ester derivative (Fura-2/AM), is commonly used to measure intracellular Ca2+. Its molecular mass is 831. Goldman equation: the Goldman– Hodgkin–Katz equation is used to calculate the reversal potential across a cell membrane based on all the ions that are permeant through the membrane. Lipopolysaccharide (LPS): a major component of the membrane of Gram-negative bacteria and a potent stimulant of innate immunity. It is also often referred to as endotoxin. Lucifer yellow: an ionic fluorescent dye commonly used to study cell morphology. It can be introduced into the cytoplasm by microinjection or P2X7R-mediated permeabilization of the plasma membrane. Its molecular mass is 444. miRNA: small noncoding RNA active in post-transcriptional regulation of gene expression. N-methyl-D-glucamine (NMDG): an organic monovalent cation often used to replace Na+ as a component of the extracellular solution in electrophysiology experiments. It

While ATP-mediated permeabilization is a common observation in P2X7R-expressing cells in vitro, whether this also occurs in vivo is unknown. In principle, it can be assumed that shortlasting pore openings can occur when P2X7-expressing cells are exposed to high extracellular ATP concentrations, such as at inflammatory or tumor sites. Such transient openings in the plasma membrane may allow direct entrance into the cytoplasm of low-molecular-mass extracellular molecules (or even of linear small peptides or miRNAs), as well as the release of cytoplasmic molecules. In this respect, the P2X7R might resemble plasma membrane pathways, such as connexins or pannexins, which are understood to allow the efflux of low-molecular-mass cytoplasmic solutes. The distinct feature of the P2X7R is its activation by an ubiquitous extracellular messenger (i.e., ATP). Given the potential serious consequences of P2X7R macropore opening, it is anticipated that, under most physiological conditions, the P2X7R should be silent, while, during inflammation, the P2X7R should be active to allow the generation and release of key inflammatory mediators, such as cytokines, reactive oxygen intermediates, and metalloproteases [17]. At inflammatory sites, sufficient ATP levels build up in the extracellular space to match the high activation threshold for P2X7R (0.3–0.5 mM) [18]. Thus, the high Kd of the P2X7R for ATP is a crucial safeguard mechanism that prevents ill-timed opening of the pore.

Non-Nucleotide Agonists at P2X7R-Mediated Pathways Activation of P2X7R-mediated signaling is characterized by a remarkable plasticity since it may depend not only on the presence of ATP, but also on non-nucleotide agonists that might function as positive allosteric modulators. There is evidence suggesting that agents such as amyloid-b [19], serum amyloid [20], and the cathelicidin LL-37 [21] activate cell responses via the P2X7R. However, it cannot be excluded that P2X7R activation following stimulation with non-nucleotide agonists is secondary to the release of ATP, which then feeds back onto P2X7R. 2

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The P2X7R macropore may also be opened by agonists acting on the cytoplasmic side. Indeed, macrophage activation by lipopolysaccharide (LPS), the paradigmatic bacterial endotoxin, involves P2X7R activation and ATP release [22]. Intracytoplasmic LPS lowers the threshold of P2X7R activation, thus sensitizing this receptor to ambient ATP concentrations [23]. P2X7R modulation by cytoplasmic factors is also suggested by the finding that Alu-RNA accumulation in the cytoplasm can activate P2X7R independently of ATP release [24]. In addition, other non-nucleotide compounds, such as the antibiotic polymixin B, can potentiate P2X7R pore formation in response to ATP stimulation [25]. P2X7R permeability can be also modulated by changing the P2X7 subunit composition. The human P2X7R is expressed in several splice variants, the canonical full-length monomer being named P2X7A [17,26]. A common widely expressed truncated isoform, named P2X7B, lacks the C-terminal 249 amino acids, and harbors 18 extra amino acids after residue 346. The receptor resulting from P2X7B monomer assembly shows ion channel activity but no macropore function [15]. However, co-expression of P2X7B together with P2X7A leads to formation of a functional P2X7A–P2X7B heterotrimeric receptor that shows enhanced macropore function compared with the homotrimeric P2X7A receptor. The heterotrimeric P2X7A–P2X7B receptor shows higher affinity for ATP and an enhanced ability to support cell energy metabolism and proliferation. Despite having been neglected for many years, P2X7R is regaining interest from immunologists and oncologists as an appealing target for immunotherapy. A potent and high-affinity inhibitory monoclonal antibody has been created [27], while extension of the nanobody technique to P2X7R has generated high-affinity reagents with potent inhibitory or alternatively stimulatory activity with potential therapeutic applications [28,29].

Pathophysiological Functions of P2X7R: Is the Macropore Implicated? P2X7R is a potent proinflammatory receptor [17,26]. Secretion of several cytokines and chemokines is dependent upon its activation, the best documented being IL-1b. Secretion of biologically active IL-1b is a two-step process whereby transcription of the[36_TD$IF]IL-1b gene (step 1) driven by Toll-like receptor (TLR) activation promotes accumulation of pro-IL-1b in the cytoplasm, followed by NLRP3 inflammasome activation and pro-IL-1b cleavage to IL-1b (step 2). In an as yet poorly understood fashion, IL-1b maturation is coupled to its release. Assembly and activation of the NLRP3 inflammasome is strongly stimulated by P2X7R activation [2]. It has been suggested that a decrease in the local cytoplasmic K+ concentration is the likely coupling messenger [30]. Thus, in principle, activation of the macropore should not be necessary for this process since ion fluxes across the P2X7R channel should suffice. However, most data show that a large drop in cytosolic K+ is required [30], suggesting that K+ efflux across the P2X7R channel might not be sufficient and, thus, postulating a pathway with higher K+ conductance. Activation of P2X7R at restricted plasma membrane sites might be able to generate a large local K+ decrease in the absence of an extensive and highly dangerous perturbation of cytoplasmic homeostasis [31]. P2X7R has been also implicated in the production of reactive oxygen species, release of lysosomal enzymes, shedding of plasma membrane metalloproteases, killing of intracellular pathogens, fusion of inflammatory macrophages to generate multinucleated giant cells, activation of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) transcription factors, shedding of plasma membrane-derived microvesicles, release of tissue factor and vascular endothelial growth factor (VEGF), phagocytosis of nonopsonized particles and bacteria, clearance of

allows measurement of pore size of ion channels. It has a molecular mass of 195. Nanobodies: single-domain antibodies based on the antigenbinding domain of heavy chain-only antibodies from camelids. NOD-like receptor containing a pyrin (NLRP3): a member of the family of NOD-Like receptors (NLR) that is the most potent activator of pro-caspase-1 cleavage and IL-1b maturation and release. Nuclear factor kappa-light-chainenhancer of activated B cells (NF-kB): a transcription factor that controls transcription of many genes involved in inflammation and cell survival. Nuclear factor of activated T cells cytoplasmic 1 (NFATc1): a component of the transcription factor NFAT, with a pivotal role in gene transcription during the immune response. Propidium: a DNA-intercalating fluorescent dye commonly used to stain dead cells because it normally does not cross the plasma membrane of live cells. It has a molecular mass of 668. Reversal potential (Vrev): the reversal potential for a particular ion is the membrane potential at which there is no net flow of that ion across the membrane. Spermidine: a polyamine compound with a range of biological functions and a molecular mass of 145. Toll-like receptors (TLRs): a family of proteins involved in innate immunity and expressed mostly by mononuclear phagocytes. They are mammalian orthologs of the proteins encoded by the gene Toll in Drosophila. Tris+: hydroxymethylaminomethane, organic cation often used to replace Na+ in electrophysiology experiments; has a molecular mass of 121. Vascular endothelial growth factor (VEGF): the most potent angiogenic factor. Yo-Pro: a carbocyanine cationic dye that binds to nucleic acid and is commonly used to stain apoptotic cells. It has a molecular mass of 629.

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CD

ATP

ATP

3 CD7

ATP

ATP

ATP

ATP ATP

P2X VRAC

ATP

ATP

P2Y

ATP ATP

ATP

Glucose ATP

Lactate/pyruvate

ATP

Pan xP2X

1

39

CD39

CD73

73

ATP

ATP

ATP

VR

ATP

CD

ATP

ATP

ATP

ATP ATP

CD

P2X

CD39

AC

ATP

P1

P1

ATP

P2X

P2X

P2Y

ATP

ATP

P2Y

X P2 x-1 n Pa

ATP

ATP

ATP

ATP

ATP

ATP

P1

CD73

P2Y

CD73

CD39

39

P1

P2Y

CD

73

P2 X

39

CD

P AT

Figure 1. ATP Is a Autocrine/Paracrine Extracellular Mediator Released via Multiple Pathways. ATP accumulates in the cell cytosol as a product of oxidative phosphorylation and aerobic/anaerobic glycolysis, and can be exported directly from the cytosol via plasma membrane channels (e.g., pannexin-1, panx-1, or volumeregulated anion channels, VRAC) or, alternatively, can be loaded into secretory vesicles (green) to be released by secretory exocytosis, or be inserted into plasma membrane-derived microvesicles (light blue). Once in the extracellular space, ATP ligates plasma membrane P2Y or P2X receptors, or is sequentially degraded to

apoptotic cells, and cancer cell proliferation (reviewed in [17]). Some of these functions (i.e., phagocytosis and transcription factor activation) do not appear to require macropore activity, while others, (i.e., killing of intracellular pathogens and formation of multinucleated giant cells) have been suggested to depend on macropore formation. Indeed, expression of the P2X7R, on the one hand, accelerates fusion of phagosomes with lysosomes to generate phagolysosomes [32,33], a key process for intracellular pathogen killing, and, on the other hand, promotes fusion of activated macrophages to generate multinucleated giant cells [34]. The role of the P2X7R macropore in membrane fusion is poorly understood, but might comprise facilitating the formation of the early fusion pore that precedes membrane fusion. Sustained opening of the P2X7R macropore triggers caspase-3 cleavage and cell death via necrosis or apoptosis, depending on the given cell type and the experimental conditions. Formation of the macropore is an absolute requirement for P2X7R-dependent lysis, because cells transfected with the defective P2X7B isoform are fully resistant to ATP-dependent cell death. The ionic composition of the incubation medium is also crucial for the cytotoxic ATP

4

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(A)

P2X7 Monomer

COOH

TM2

5’

Exon 5

Exon 9

1568 N

H521 Q

E496 A

Q46 0R

T357 S

A348 T

TM1 R307 Q

H1 55Y

NH2

Exon 11 Exon 13 Chromosome 12q24.31

3’

(B)

P2X7 Trimer YO-PRO-1 Na+ Ca2+

Lucifer yellow Na+ Ca2+ ATP

K+ K+

ATP ATP

Closed

ATP

Open

Figure 2. Structure of the P2X7 Subunit and Hypothetical Mechanism of Formation of the Macropore. (A) The P2X7 subunit is a 595 amino acid-long protein that spans the plasma membrane with two a-helical domains. It also has a bulky extracellular region and N- and C-terminal residues both on the cytoplasmic side. The shape of the 3D resolved structure of the P2X7 subunit has been likened to a ‘dolphin’, where the fins represent the transmembrane TM1 and TM2 a-helices. Several single-nucleotide polymorphisms (SNPs) have been identified, some of which are highlighted in blue (gain-of-function), yellow (loss-of-function), or cyan (neutral). Their position relative to the plasma membrane is also shown. (B) Assembly of P2X7 subunits to form the functional homotrimeric P2X7 receptor (P2X7R) is shown. Binding of three ATP molecules gates a channel/pore that allows the influx of monovalent cations and fluorescent dyes, such as Yo-Pro. It is not clear how large anionic dyes, such as Lucifer yellow, permeate through the plasma membrane; thus, participation of an accessory molecule cannot be excluded. The photograph in (B) shows a pattern of diffuse Lucifer yellow staining in mouse

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effect since incubation in low-salt medium (i.e., 250 mM sucrose) delays ATP-stimulated cytotoxicity by several hours despite allowing full plasma membrane permeabilization. Rather paradoxically, basal expression of P2X7R supports cell survival and proliferation [35]. Such a trophic effect is dependent on the potentiation of oxidative phosphorylation, which in turn promotes a striking increase in total cellular ATP content [36]. A trophic effect has also been described in cells transfected with P2X7B; thus, macropore function is likely not needed to support energy metabolism. Finally, while basal P2X7R expression supports mitochondrial activity, sustained P2X7R activation precipitates a mitochondrial catastrophe that ends rapidly in cell death [37].

Molecular Basis of the P2X7R Macropore: Where Electrophysiology and Cell Biology May Disagree Ligand-triggered movements of the TM2 domains of P2X7 subunits open the ion permeation pathway leading to Na+ and Ca2+ influx and K+ efflux. During prolonged (i.e., several seconds) application of ATP, ion fluxes are followed (or paralleled) by uptake of water-soluble fluorescent markers [7,13,38,39]. This observation inspired the hypothesis that macropores are formed in addition to the fast opening of the P2X7R ion channel pore explored by electrophysiology, but they have since remained elusive. Two mechanisms have been proposed to explain macropore formation: (i) progressive dilatation of the P2X7R ion channel pore; and (ii) recruitment of an accessory pore-forming molecule downstream of P2X7R activation [40,41]. Voltage clampbased measurements of ionic currents were extensively used to record the earliest steps of P2XR activation and to directly measure the ion conductance of the ATP-induced pores. P2X7R-Mediated Whole-Cell Currents Measurement of the net ion current in the whole-cell configuration demonstrated that the time course of the ion flow through activated P2X7Rs during even the first seconds of application of ATP or the often-used more specific P2X7R agonist benzoyl ATP largely depends on the experimental conditions. Simple exponentially saturating [42], approximately linear [43], supralinear [44], and inactivating [45,46] time courses of P2X7R-dependent current activation have been reported. Often, two different current components have been observed: an early, ultrafast component developing within a few milliseconds (often called I 1), followed by a second component (I 2), which develops within seconds [47]. Accordingly, repeated applications of P2X7R agonists were reported to evoke currents of equal [42], successively increasing [43,48,49], or decreasing amplitude [45,46]. These divergent shapes of ion currents may point to completely different P2X7R activation mechanisms. However, more probably, the involvement of other downstream-activated ion conductances may contribute to the dissimilar currents. Electrophysiological evidence for macropore formation was seen in the shift of reversal potential of ramp currents measured in extracellular solutions with Na+[367_TD$IF] substituted by large cations [e.g., N-methyl-D-glucamine (NMDG+)] [47–49]. Since P2X7R channels are initially permeable to small cations, such as Na+ and K+, the replacement of extracellular Na+ by larger cations, such as NMDG+ or Tris+, shifts the reversal potential Vrev of P2X7R currents to negative values according to the Goldman equation. As observed, when a pore dilatation occurs, the accompanying increased permeability to organic cations shifts Vrev back to less negative values, thus strengthening the concept of ion channel pore dilatation. At odds with this conclusion, this backwards shift of Vrev was associated with a reduced ion conductance in most cases [47,48,50,51]. This controversy was resolved recently when it was shown that long-lasting activation of another member of the P2X receptor family, P2X2R, leads to accumulation of the large organic cations in the voltage-clamped cell, causing the following 6

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shift of Vrev towards more positive potentials [10]. Earlier investigations had already pointed to this explanation, where the Vrev shift was observed only if, during the long-lasting benzoyl ATP applications, Na+ was exchanged for NMDG+ throughout the experiment. If, however, Na+ was substituted by NMDG+ only intermittently, the Vrev shift did not occur [50]. In the case of P2X7Rs, which have an intrinsic low but measurable conductance to NMDG+, this accumulation-dependent phenomenon is enhanced by a drastic increase in the open probability of P2X7R channels due to extracellular Na+ removal [12,52]. This means that the often measured Vrev shift can no longer be taken as evidence for P2X7R pore dilatation. Misinterpretation of whole-cell voltage data might have been fueled not only by unaccountedfor ion accumulation, but also by contamination of the ATP-induced current by other, not directly P2X7R-mediated, currents. Changes in intracellular ion concentrations may not only disturb Vrev measurements of the pure P2X7R-mediated current, but also induce other ion conductances downstream of P2X7R activation. In particular, an increase in the intracellular Ca2+ concentration may directly activate Cl channels [53]. Furthermore, P2X7-dependent activation may lead to depolarization of the mitochondrial membrane and, if protracted, to mitochondrial dysfunction [37], inhibition of ATP production, and cellular demise. P2X7Rmediated apoptosis may be paralleled by opening of pannexin-1 hemichannels [54]; furthermore, ischemic-like conditions may open connexin hemichannels [55]. In unclamped cells, even the long-lasting depolarization induced by P2X7R channel opening may activate a large unspecific conductance, and P2X7R-driven cell swelling [56] may activate volume-dependent anion channels, which may release ATP [57], an event often observed upon P2X7 receptor activation [58,59]. Therefore, changes in the intracellular ion composition due to P2X7R activation-dependent ion fluxes may serve as second messengers activating downstream [369_TD$IF]conductances. Indeed, the potentiation of P2X7R-dependent currents [14] and the permeabilization to large molecules [60,61] induced by long-lasting agonist application depend on the density of P2X7Rs expressed on the cell membrane. For example, human B lymphocytes, which normally express P2X7Rs at a low level, display unchanged P2X7R-dependent ion currents with repeated ATP applications [42]. Furthermore, Ca2+[368_TD$IF] and ethidium uptake by ATP-stimulated B lymphocytes is enhanced by the removal of extracellular Na+ [39,62,63], which potentiates P2X7R activation. Another interpretation of the dependence of the pore formation on the expression level might be that P2X7R clustering is necessary for pore formation. Although an early study discarded this possibility [64], more recent data leave this mechanism open [31]. P2X7R-Mediated Single-Channel Currents Complications intrinsic to the whole-cell voltage clamp measurements can be circumvented to a large extent by patch clamp analysis, where activated single P2X7R channels can be observed [65]. This method not only avoids the spoiling of P2X7R-dependent currents, but also enables direct observation of the effects of modulators on P2X7R ion channel conductance and open probability. In oocytes heterologously expressing human P2X7R, cationic channels with a conductance of approximately 10 pS and a pore diameter of 8.5 Å were gated by ATP4–[370_TD$IF] within a few ms [12]. ATP-activated channels of similar conductance were earlier recorded in macrophages [66], mast cells [67], and B lymphocytes [68]. The removal of either extracellular Na+, Cl , or H+ increased the open probability of the P2X7R channel by prolonging the time the channel spends in the open state (Na+ removal [12]) or shortening the closed-state dwell times (Cl [69] or H+ removal [70]). Furthermore, single-channel current measurements demonstrated that human P2X7R is not anion permeable [69].

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None of the single-channel recordings support the concept of pore formation due to dilatation of the P2X7R channel ion pathway. During long-lasting activation of single P2X7Rs, the single channel current amplitude and the permeation characteristics remained constant [12], even under experimental conditions where the open probability of the P2X7 channel was high due to Na+ removal or mutations in the TM2 region [71]. Gating and permeation of human P2X7R remained stable for several minutes (as long as the patch was intact), and were largely dependent on the Ser342 residue in the TM2 [71]. Stable P2X7R-dependent currents could also be evoked in patches containing more than one channel [12]. These single-channel features measured in patches excised from Xenopus oocytes heterologously expressing human P2X7R did not differ from those recorded either in the cell-attached configuration, where the cytoplasmic environment of the channel is largely retained [12], or from patch clamp measurements of single human P2X7R channels performed in B lymphocytes [68]. The single-channel data, the finding that some P2X7R-expressing cells do not display macropore formation [43,62], the observation that pore formation is more temperature dependent than ion channel gating [7,44], and the finding that the P2X7R channel does not undergo dilatation, all suggest that additional mechanisms must underlie the opening of the macropore.

Mechanism of Macropore Formation There appears to be an asymmetry between cell biology and electrophysiology in the description of P2X7R permeability properties. The first evidence of permeation behavior of P2X7R (originally ‘the ATP4– receptor’ and later P2Z) came from cell biology experiments [6,72]. Only later was pore formation investigated by electrophysiology [66,67]. Challenge with ATP of rat mast cells, mouse, and human macrophages, or HEK293 cells transfected with human or rat P2X7R, triggered uptake of Ca2+ (for which pore formation is not necessary) as well as of Lucifer yellow, Yo-Pro, propidium, or ethidium [7,9,72]. In most experiments, ATP concentration-dependencies of Ca2+ influx and fluorescent marker uptake are indistinguishable; however, the kinetics of fluorescent dye uptake is slower. This has inspired the ‘two-step’ or the ‘channel-to-pore transition’ model to explain the permeability increase to fluorescent dyes. However, this model might have been the result of a mistaken interpretation of the dye uptake kinetics, because the fluorescence increase due to dye uptake is slower than the fluorescence increase produced by Ca2+-sensitive dyes following Ca2+ influx. Therefore, it might be that macropore formation occurs at the same time as the opening of the cation-selective channel (i.e., no channel-to-pore transition occurs) and the Ca2+ influx is detected before fluorescent dye uptake simply because the two signals develop over different timescales. Recent electrophysiological analysis clearly demonstrated that small (Ca2+ or Na+) and larger cations (NMDG+ or spermidine) permeate without lag through the P2X7R channel [52,71]. This view is supported by experiments investigating the permeability properties of receptors formed by the assembly of mutated or truncated P2X7 subunits: truncations or mutations that abrogate uptake of fluorescent markers also drastically decrease cation fluxes [14,15,73]. Recent investigations demonstrated that the P2X7R pore may reach a diameter of 8.5 Å [12], which is compatible with the permeation of the cationic dye Yo-Pro (7  8  19 Å) [16], suggesting that cationic dyes of similar size directly permeate through P2X7R. This finding aligns with previous data showing that the P2X7R channel allows entry of nanometer-sized cationic and even of anionic molecules (Table 1) [38]. However, several other findings argue against a function of P2X7R as an all-round pathway for organic molecules. The biphasic increase of P2X7R-dependent ion currents [47,74], the observation that, in contrast to the P2X7R-dependent ion currents, the dye uptake often needs temperatures >20  C [7,75], the fact that the cation selectivity of single human P2X7R [12] should exclude the permeation of 8

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Table 1. Molecules Reported to Permeate through the P2X7R Pore Compound

MW

Diameter (Å)a

Charge

In/outb

Physiological/experimental

Refs

Na

23

1.2

+

In

Physiological

[9,12]

K

39

1.5

+

out

Physiological

[12]

Ca

40

1.4

2+

In

Physiological

[9,62]

Tetramethylammonium

74

6.7

+

In

Experimental

[12]

Tris [358_TD$IF]

121

7.4

+

In

Experimental

[12]

Spermidine

145

3

3+

out

Physiological

[52]

MTSEA

156

5.5

+

In

Experimental

[38,71]

NMDG

195

7.2

+

In

Experimental

[9,12]

+

Ethidium

394

11

+

In

Experimental

[38]

Yo-Pro

629

8

2+

In

Experimental

[9]

Propidium

668

11.5

2+

In

Experimental

[87]

SCNc

97

4.3

–

In

Experimental

[88]

380

9

4–

In

Experimental

[13]

387

11

–

In

Experimental

[38]

444

13

2–

In

Experimental

[7]

507

12

4–

out

Physiological

[59]

Fura-2 free acid

831

16.5

5–

In

Experimental

[7,80]

Doxorubicin

543

32

Uncharged

In

Experimental

[89]

EGTA

c

FITCc Lucifer yellow

c

ATPc c

a

The diameters are given for the nonhydrated ions. The direction of flux depends on the electrochemical force, (i.e., the concentration gradient of the chemical species, the charge and the membrane potential). c The P2X7R-dependent increase in plasma membrane permeability for anions is not settled. It cannot be excluded that P2X7R activates two separate permeation pathways, one for cationic and one for anionic dyes with different activation properties. b

Table 2. Evidence Supporting or Disproving the Hypothesis that Macropore Formation Requires an Accessory Molecule Extrinsic to P2X7R Supporting

Disproving

P2X7 expression in Xenopus oocytes was never shown to induce ATPstimulated fluorescent dye uptake [45]

P2X7 expression in many different mammalian eukaryotic cells induces ATPdependent fluorescent dye uptake with high reproducibility [17]

Electrophysiological analysis describes a cation-selective channel with little or no permeability for anions (the anionic dye Lucifer yellow should not be admitted) [9,12,69,71]

Cells from P2X7-deleted mice lack both ion channel and macropore activity [17,91]

Electrophysiological analysis and cysteine scanning set an upper limit for the diameter of permeating molecules of 8.5 Å, too small for ethidium and propidium [12,71]

The two most likely candidates for the role of P2X7R accessory proteins, pannexin-1 and connexin-43, were shown to be dispensable for P2X7Rdependent macropore formation [78,79]

Ion fluxes and fluorescent dye uptake have substantially different temperature sensitivities [7,44]

Single amino acid mutations in TM2 affect both channel and macropore functions [38]

Single amino acid replacements may impair fluorescent dye uptake without affecting ion fluxes [76,77]

P2X7R reconstituted into artificial liposomes generates a nonselective pore permeable to Yo-Pro [16]

Some cell types display ion channel activity but no macropore formation [43,62,92]

Ca2+[359_TD$IF] (as measured with fluorescent dyes) and ethidium bromide uptake show overlapping ATP dose-dependency curves [93,94]

The Vrev shift to more positive potentials taken as evidence for P2X7R channel dilatation and, therefore, macropore formation, is an artifact due to the cytoplasmic accumulation of positively charged large molecules [10,50]

P2X7 truncations that hamper or even abrogate macropore formation also reduce channel function [15,95]

K+ and Na+ fluxes are detected before efflux of radiolabeled intracellular nucleotides [96]

P2X7-dependent nonexocytotic glutamate release from neurons does not require accessory-pore-forming molecules [95]

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anionic dyes, the different characteristics of cationic and anionic dye uptake [75], and evidence that mutations in the C-terminal region may abolish the uptake of fluorescent dyes [76] but have no influence on the ion channel function of the P2X7R [77], all point to additional pathways (Table 2). However, candidates proposed so far (connexin 43, P-glycoprotein, and especially pannexin-1) have not survived experimental testing [78,79]. An uptake of fluorescent probes via endocytosis secondary to P2X7R stimulation can be ruled out since it has been repeatedly shown that, soon after P2X7R activation, Lucifer yellow or other nonpermeant dyes, such as Fura-2 free acid, are homogeneously and diffusely localized in the cytoplasm [7,80]. Thus, assuming that a P2X7R-associated accessory molecule responsible for macropore formation exists, we have no clue as to its identity. This also holds for the 400 pS channels, which are indirectly activated by P2X7R [7,13,81]. Assuming that a dye uptake pathway of sufficient size to allow Yo-Pro uptake is intrinsic to P2X7R, a recent report suggests both a molecular mechanism by which pore size can be modulated and how the long P2X7 COOH-terminal tail participates in this process [16] (Figure 3). Yo-Pro uptake could be induced via P2X7R incorporated into artificial liposomes

P2X7 Receptor

P2X7 Receptor

YO-PRO-1

C363

TM2

C362 COOH

COOH C362 C370

TM2

TM2

TM2

ATP

COOH

C363

COOH

C370

Cysteine palmitoylaon (Cys-rich region CRR)

Figure 3. Hypothetical Mechanism of Modulation of P2X7 Receptor (P2X7R) Permeability by Removal of Cholesterol Inhibition. Cholesterol (yellow chain) is proposed to interact with TM2 and inhibit P2X7R-associated permeability increases, preventing formation of the macropore and influx of fluorescent dyes. The P2X7 C-terminal tail harbors cysteine residues (green stars) that can be palmitoylated. Palmitoylation enhances the interaction of the COOH tail with the plasma

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(i.e., as the sole protein present). Dye uptake was strictly dependent on lipid composition of the membrane, because it was facilitated by phosphatidylglycerol and sphingomyelin and inhibited by cholesterol directly interacting with P2X7R at TM2. It has been suggested that the COOH tail facilitates macropore opening because it bends and inserts into the plasma membrane via the presence of palmitoylated cysteine residues, thus preventing cholesterol interaction with the P2X7R. Accordingly, truncated P2X7 isoforms are unable to form the macropore since they lack the ‘protective’ effect of the COOH tail [16]. A facilitating effect of cholesterol depletion on P2X7R pore formation was previously reported [82,83], while another finding showed that P2X7R is associated with detergent-resistant membranes (cholesterol rafts), and is palmitoylated in the COOH tail [84]. Modulation of P2X7R permeability by cholesterol might help explain the mechanism by which the non-nucleotide P2X7R agonist LL-37 restores macropore formation by the truncated P2X7DC receptor. The LL-37 cathelicidin is repelled by cholesterolrich membranes [85]; therefore, it can be hypothesized that it promotes P2X7R release from rafts, thus facilitating macropore formation. P2X7R-dependent redistribution of the phospholipid phosphatidylserine, acting at the level of inhibition of the multidrug transporter p-glycoprotein, was implicated in the mechanism underlying organic molecule uptake, and also as a feed-forward stimulus for P2X7R cation channel activation, but there have been no further reports supporting these initial findings [86].

Outstanding Questions Do pathways for uptake of cationic (e. g., spermidine or Yo-Pro) and anionic hydrophilic molecules coincide? Does the P2X7R pore allow transmembrane fluxes of other biologically relevant molecules, such as miRNAs? Is the plasma membrane cholesterol and phospholipid composition a main factor dictating the different P2X7R permeability features observed in different cell types? Based on the dramatic effect of plasma membrane cholesterol content on P2X7R permeability, is it possible that other factors (e.g., non-nucleotide agonists) trigger macropore opening simply by changing the cholesterol content of the P2X7R membrane microenvironment?

Concluding Remarks About 40 years after the first detailed descriptions of ATP-induced changes in plasma membrane permeability [6,72], we are now provided with a reasonable interpretation of the molecular mechanism responsible. Convergent evidence from the two most commonly used techniques to investigate this process (electrophysiology and fluorescent dye uptake) gives concordant answers: (i) no channel-to-pore transition (i.e., no pore dilatation) occurs during receptor activation; (ii) P2X7R-dependent permeability to Na+, Ca2+, K+, and higher-molecularmass organic molecules, such as NMDG+ or spermidine3+, develops within the same time course; and (iii) the pathway for uptake of the fluorescent dye Yo-Pro is likely to be intrinsic to the P2X7R, and no accessory molecules are needed. Nevertheless, other P2X7R-mediated permeability pathways may exist, but their identity and physiological role remain to be established (see Outstanding Questions). Clarification of the mechanism underlying the most striking (and possibly unique) function associated with such a relevant receptor in inflammation and immunity will certainly have important implications in biology and medicine.

Might an increased plasma membrane cholesterol content explain the refractoriness of some cell types to ATPmediated permeabilization and, therefore, to ATP-stimulated cell death? Since the P2X7R is highly expressed in tumor cells, could the P2X7R macropore be exploited to enhance cellular loading of chemotherapies? Is the flux of large organic cations through the P2X7R in vivo large enough to be physiologically and pathophysiologically relevant?

Acknowledgments F.[372_TD$IF]M. and G.S. are supported by grants from the Deutsche Forschungsgemeinschaft (Ma1581/15-3; Schm536/9-3). F.D. V. is supported by grants from the Italian Association for Cancer Research (n. IG 13025 and IG 18581), the Ministry of Health of Italy (n. RF-2011-02348435), and institutional funds from the University of Ferrara. The authors wish to thank [37_TD$IF]Dr. Alba Clara Sarti for invaluable help and advice. We apologize to the authors of many excellent articles on this topic that we were unable to cite due to constraints in the number of allowed references.

[374_TD$IF]Disclaimer Statement F.D.V. is a Member of the Scientific Advisory Board of Biosceptre, a UK-based Biotech Company[375_TD$IF]. G.S. and F.M. declare no conflict of interest.

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