Assembly and trafficking of P2X purinergic receptors (Review)

Abstract

P2X receptors are cation selective ion channels gated by the binding of extracellular ATP. Seven subtypes have been identified and they have widespread and overlapping distributions throughout the body. They form homo- and heterotrimeric complexes that differ in their functional properties and subcellular localization. They form part of larger signalling complexes, interacting with unrelated ion channels and other membrane and cytosolic proteins. Up- or down-regulation of their expression is associated with several disease states. This review aims to summarize recent work on the assembly and trafficking of this family of receptors.

• P2X purinergic receptors
• assembly
• trafficking

Introduction

Extracellular ATP is an important signalling molecule that controls numerous biological processes in the nervous, immune, endocrine, respiratory and cardiovascular systems [1–4]. Its effects are mediated via cell surface P2 receptors of which there are two types: the G protein coupled P2Y receptors and the P2X receptors, which possess an integral cation channel that opens in response to ATP. The P2X receptors represent important therapeutic targets in pain, inflammation and stroke [4], [5]. Seven members of this family, sharing 35–50% amino acid identity, have been identified in vertebrates [6]. These subunits show a widespread and overlapping distribution and are able to coassemble as heteromeric receptors with differing pharmacological properties [7]. Unrelated proteins such as nicotinic and 5-HT receptors and pannexin hemichannels also appear capable of interacting with P2X receptor subunits. The response of P2X receptors is further regulated by controlling the trafficking of receptors to the plasma membrane and intracellular compartments, by the interaction between motifs in specific subtypes and additional accessory proteins.

Physiological roles of P2X receptors

The physiological roles of these receptors have been recently reviewed [1–3] and a summary of what has been confirmed using mice with P2X receptor genes either knocked out or knocked down is provided in Table I in Supplementary data (online version only).

Figure 1.  Trafficking of P2X receptors to and from the plasma membrane. The P2X receptor subtypes are broadly classified into four groups based upon their trafficking behaviour. Motifs that have been shown to be determinants of receptor trafficking are indicated. Plasma membrane (1–2), P2X_1-7 receptors stabilized by YXXXK motif. Deletions within LPS-binding motif of P2X₇, inhibit plasma membrane expression. Endosomes (4–6), P2X₄ constitutively internalized from the plasma membrane via dynamin and AP-2 dependent process involving IL and YXXGL motifs. Receptors can be recycled but are predominantly targeted to lysosomes. P2X_1,5 and ₇ are internalized in response to chronic agonist application. They recycle back to the plasma membrane. Lysosomes (3, 7–9), P2X₄ is targeted to lysosomes, protected from degradation and released via exocytosis. P2X_4/6 and P2X_4/5 assemblies are also targeted here. Delivery to lysosomes may be via a direct pathway from the TGN as well as an indirect pathway via the plasma membrane. ER, P2X_1,2,3,5 exported as homo- and heterotrimers. P2X₄ rapidly exported as homo- and hetero-trimers. P2X₆ retained as a monomer or exported as a heterotrimer. P2X₇ exported as a trimer or retained incorrectly assembled.

Molecular nature of P2X receptors

The P2X receptors belong to a different structural class to the Cys-loop and glutamate receptor families of ligand gated cation channels [8–10]. Each subunit is between 379–595 amino acids in length, contains two transmembrane (TM) segments, and between 50–70% is contributed by a large extracellular loop that contains 10 disulfide bonds and several N-linked glycosylation sites. The N- and C-termini are cytoplasmic and, in the case of P2X_{1, 3, 4, 5} and ₆, they are both short. P2X₂ has a longer C-terminus (120 amino acids) that is involved in interactions with other ligand gated ion channels of the Cys-loop family. P2X₇ has a 240 amino acid C-terminal domain that is similarly involved in interactions with other proteins, suggesting that this receptor forms part of a multi-protein signaling complex. As yet, no high-resolution structural information is available for P2X receptors, however this information has recently been obtained for an acid sensing ion channel (ASIC) that shares a similar trimeric architecture with members of the P2X family [11]. Although there is no obvious sequence similarity between these families, in both cases the subunits possess two TM regions, a large extracellular domain of similar length containing multiple disulphide bonds and a pore primarily lined by residues from TM2. The ASIC structure might therefore prove to be a useful model to make predictions about P2X receptor structure-function relationships.

Evidence for trimeric complexes

P2X receptors are multimeric proteins. The first direct evidence for this was the demonstration that P2X₂ and P2X₃ can coassemble to produce heteromeric channels with functional properties that distinguish them from the parent homomeric receptors [12]. For the majority of multimeric integral membrane proteins, assembly takes place at the endoplasmic reticulum (ER) membrane, and a quality control machinery ensures that only the correctly folded and assembled complexes are exported from the ER. A key role in this process is played by the processing of core glycans that are added at consensus Asn-X-Ser/Thr motifs as soon as the nascent polypeptide chains enter the ER [13].

Several different approaches have been used to determine the stoichiometry of P2X receptors; there is now a consensus that these receptors are trimeric. For many years, however, the issue of stoichiometry was controversial because of a report that when the large extracellular domain of the P2X₂ subunit was expressed in Escherichia coli in isolation from the rest of the protein, it assembled to form stable tetramers [14]. These assemblies bound agonist (ATP) and antagonist (suramin), suggesting that they closely resembled the arrangement of the extracellular domains of the intact receptor complex. This now seems unlikely to be the case, and there is evidence to support a role for the transmembrane segments as important determinants of subunit coassembly [15].

The first study to provide evidence for the trimeric structure of recombinant P2X receptors was by Nicke et al. [16]. They purified His-tagged P2X₁ and P2X₃ receptors expressed in Xenopus oocytes, cross-linked the complexes using bifunctional analogues of the antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), and analyzed their size by SDS-PAGE. For both receptors, this revealed a single protein band corresponding to a homotrimer and similar results were obtained when the receptor complexes were resolved under non-denaturing conditions. A later study from the same group demonstrated that P2X₂, P2X₄ and P2X₅ similarly form stable homotrimers [17]. In addition to trimers, higher order complexes were also resolved with molecular weights consistent with hexameric and nonameric structures. The stability of these structures was dependent upon the detergent used for solubilization and, unlike the trimers, they were not cross-linked using the short-arm linker glutaraldehyde. This suggests that trimeric assemblies are able to form stable clusters, but subunits belonging to different trimers are further apart within the cluster than subunits within a trimer.

Visualization of individual P2X₂ receptors by atomic force microscopy (AFM) has also provided direct evidence for the trimeric nature of these complexes. Barrera et al. [18] imaged receptors adsorbed to poly-L-lysine coated mica and from the particle dimensions they calculated the molecular volume to be 409 nm³, close to that expected for a receptor trimer (473 nm³). In addition, they decorated particles with bound monoclonal anti-His IgG and measured the angle separating two bound antibodies as 123°, very close to the value of 120° predicted for a trimeric receptor. AFM was also used to image purified P2X₂ receptors in the aqueous phase [19] and low resolution images of P2X₂ receptors obtained using electron microscopy showed structures with a triangular shape [20].

Unlike other members of the P2X receptor family, P2X₆ appears not to form stable homotrimers and as a result it is retained in the ER by the quality control machinery [17], [21], [22]. A comparison of P2X₆ and P2X₂ receptor volumes by AFM showed that the volume of P2X₆ particles (145 nm³) was a third of that of P2X₂ particles, consistent with it being a monomer [18]. Deleting the uncharged first 14 amino acids of P2X₆ or substituting positive or negatively charged residues for serines at positions 3 and 11 was sufficient to increase surface expression of the receptor [23]. The double S3K, S11K mutation was also shown to stabilize the formation of P2X₆ homotrimers as measured by the appearance of a second peak in the frequency distribution of molecular volumes. This result suggests that the N-terminus of P2X₆ is normally engaged in a hydrophobic interaction which destabilizes assembly of homomeric complexes.

Mechanisms of assembly

P2X receptors assemble in a subunit specific manner and yet little is known about the mechanisms controlling this specificity. No single domain has been identified as the determinant for the compatibility of interactions between subunits. The high resolution structure of the ASIC channel shows extensive subunit-subunit interactions within the trimeric complex, particularly involving a β-sheet region within the ectodomains and both TM domains. The picture that emerges from analysis of deletion mutants of P2X receptors is consistent with this; these studies point to the involvement of TM and extracellular regions but provide no evidence to support a crucial role for N- and C-terminal cytoplasmic regions [21]. There may be differences between the subtypes, for example for P2X₂, a mutant lacking the entire TM1 and N-terminal regions co-immunoprecipiated with the full length subunit whereas a mutant that terminated 25 amino acids upstream of TM2 did not, suggesting that TM2 plays a critical role in assembly [15]. In contrast a P2X₇ receptor variant lacking the entire C-terminus, TM2 and a distal part of the ectodomain, was able to co-immuonoprecipitate (co-IP) with the full length P2X₇ subunit and also to act in a dominant negative manner to inhibit P2X₇-receptor mediated responses [24]. These deletion mutants are, however, likely to be imperfectly folded making it difficult to raise firm conclusions on the molecular determinants of subunit assembly.

A splice variant of P2X₅ which lacks the C-terminal end of the ectodomain and the outer half of TM2 has a severe assembly defect and is retained within the ER [25]. Insertion of a polyalanine region at this position was sufficient to complement the missing outer portion of TM2 and restore homotrimeric formation and the export of complexes to the cell surface. This argues against the outer portion of TM2 being an important determinant of subunit compatibility and suggests that TM2 is primarily important for tethering the end of the ectodomain to the membrane and restricting its mobility, thus enabling the ER luminal region to engage in intersubunit interactions. Systematic mutagenesis of TM2 indicates only one position, a conserved asparate at position 355, where hydrophobic substitutions are not well tolerated with regards to assembly and ER export of the receptor. This side chain might engage in specific salt-bridge interactions and a possible partner is arginine at position 34 in TM1, which is also conserved in all subtypes except P2X₇.

Heteromeric P2X receptor assemblies

The formation of heteromeric P2X receptor complexes alters receptor function, pharmacology and trafficking, thereby increasing the diversity of channels expressed. This has important implications for understanding the physiological roles and the design of therapeutics targeting these receptors. There are many examples in the literature where multiple P2X receptor subtypes have been shown to be endogenously coexpressed and it therefore seems likely that heteromers are of physiological importance. The vast majority of studies that have analysed the properties of heteromeric receptors have, however, been carried out on receptors overexpressed in either Xenopus oocytes or mammalian cell lines. Thus although there are many examples where structural and functional interactions between different subtypes have been shown, it is not known whether the heteromers form predominantly under physiological settings. Below we have summarized the evidence in support of different heteromeric receptors and grouped them according to the nature of the evidence. We have placed the P2X_2/3 heteromer in a group of its own because this is the only combination that has clearly been shown to be physiologically relevant. The second group is where there is direct evidence that the two subunits can coassemble around a common conduction pore but this evidence exists only for heterologously over-expressed receptors and this includes P2X_2/6, P2X_1/2 and P2X_1/4. Finally we have subunit combinations for which there is co-IP and functional evidence for an interaction but the formation of heterotrimeric complexes has not been shown directly. For these it is possible that the interaction is between homotrimers rather than within heterotrimers. This includes P2X_4/5, P2X_4/6, P2X_4/7 and P2X_1/5 and of these, only for P2X_4/7 has an interaction been demonstrated for native receptors.

Group 1

P2X_2/3 heteromers

The P2X_2/3 heteromeric receptor currents are easily distinguishable, by their pharmacology and kinetics, from the parent homomeric receptor currents, and this property has facilitated the identification of a native correlate [12]. P2X₂ receptors are slowly desensitizing and insensitive to the agonist α,β,methylene ATP, whereas P2X₃ are rapidly desensitizing and activated by α,β,methylene ATP. When coexpressed, they produce a slowly desensitizing current activated by α,β,methylene ATP which cannot be attributed to the sum of the homomeric receptor currents. P2X₃ has the most restricted distribution of any of the P2X subtypes, but is found in a subpopulation of sensory neurones together with P2X₂ [26]. Currents with similar properties to those of the heteromeric receptor have been recorded from nodose, coeliac, superior cervical and dorsal root ganglia neurones and these responses are altered in the P2X₂, P2X₃ and P2X_2/3 double knock out mice [27–29]. Further evidence supporting the existence of P2X_2/3 heteromers comes from the co-IP of the heterologously expressed subunits and the use of site directed mutagenesis combined with functional and biochemical analyses to show that coexpression of wild type P2X₃ can rescue non-functional and trafficking impaired P2X₂ mutants [30], [31]. The arrangement of subunits within the heteromeric complex was probed by introducing cysteines at positions where they can form disulphide bonds between neighbouring subunits; this approach revealed that heterotrimers have the composition P2X₂(P2X₃)₂ [32].

Group 2

(1) P2X_2/6 heteromers

P2X₂, P2X₄ and P2X₆ are coexpressed in several regions within the central nervous system and all have been shown, by immunogold labeling, to be targeted to a similar location on dendritic spines at the periphery of the postsynaptic specializations [33]. The mechanism involved in the specific targeting of these complexes is unknown; however their colocalization suggests that the native receptors in the brain are likely to be heteromeric assemblies of two or more of these subunits. P2X₆ co-IPs with P2X₂ when they are heterologously coexpressed [21] and is no longer retained in the ER but traffics to the plasma membrane [23]. Functional analysis of coexpressed receptors in Xenopus oocytes suggests that the heteromeric P2X_2/6 receptor currents are difficult to distinguish from homomeric P2X₂ receptor currents, although the incorporation of P2X₆ increases the calcium permeability of P2X₂ receptors by ∼50%, an effect that might be important for a role in synaptic plasticity [34–36]. The subunit arrangement of P2X_2/6 heteromers was determined by AFM imaging of antibody-decorated receptors purified from HEK 293 cells. Unlike P2X_2/3 receptors, both P2X₂(P2X₆)₂ and (P2X₂)₂P2X₆ complexes were observed at frequencies dependent upon the relative levels of expression of the two subunits [37].

(2) P2X_1/2 heteromers

Evidence in support of this combination has been obtained using the Xenopus oocytes expression system. An additional phenotype was described after their coexpression, with novel pH sensitivity that could not be attributed to the homomeric receptor currents [38]. Blue native (BN)-PAGE analysis of purified plasma membrane receptors identified P2X_1/2 heterotrimers but not P2X₁ homotrimers suggesting that heteromer formation is preferred [17]. A physiological role for these heteromers is unknown and there is little overlap in their tissue distribution.

(3) P2X_1/4 heteromers

P2X₄ is the most widely expressed subtype and is present in most tissues that have so far been analyzed. It overlaps with P2X₁ in smooth muscle, microglia and peripheral immune cells [39], and although these two subtypes were originally shown not to co-IP when coexpressed in HEK 293 cells [21], a more recent study using Xenopus oocytes, showed that when heterologously coexpressed they form part of the same trimeric complex and that currents with a novel functional phenotype are produced. P2X₄ produces almost no response to 10 µM α,β,methylene ATP, but with P2X₁, mediates a current in response to this agonist that desensitizes more slowly than the P2X₁ homomers [40].

Group 3

(1) P2X_1/5 heteromers

P2X₁ and P2X₅ are coexpressed in the ventral horn of the spinal cord and co-IP when coexpressed in HEK 293 cells [41], [42]. P2X₁ shares similar functional properties to P2X₃ in that it produces rapidly desensitizing currents in response to α,β,methylene ATP, whereas P2X₅ is similar to P2X₂ and is insensitive to this agonist. When coexpressed, the currents show biphasic desensitization to α,β, methylene ATP and most notably, resensitize much more rapidly than the P2X₁ homomers [41], [43].

(2) P2X_4/5 heteromers

There has been no functional characterization of this combination, however they have been shown to co-IP when coexpressed in HEK 293 cells [21] and the trafficking of P2X₅ is altered by coexpression with P2X₄, such that P2X₅ colocalizes with P2X₄ in lysosomes (RML unpublished results).

(3) P2X_4/6 heteromers

P2X₄ is reported to overlap with P2X₆ in the CNS and the periphery, including endothelial cells. When heterologously coexpressed they co-IP and the distribution of P2X₆ changes such that it is no longer retained within the ER but follows P2X₄ to endolysosomes [21], [22]. Novel pharmacological properties of the heteromeric receptor currents have been reported in Xenopus oocytes [34], and this mainly involves an increase in the sensitivity of the current to antagonists such as suramin and Reactive Blue 2.

(4) P2X_4/7 heteromers

Until recently P2X₇ was thought to be unique in not forming functional heteromeric receptors with any other member of the P2X family. It is, however, coexpressed with P2X₄ in immune, endothelial and epithelial cells, and shares greater sequence similarity with P2X₄ compared with the other subtypes. P2X₇ was recently shown to co-IP with P2X₄ when heterologously coexpressed, and the endogenous receptors also co-IP from mouse bone marrow derived macrophages [44]. Coexpression of a dominant negative mutant of P2X₄ with P2X₇ reduced P2X₇ receptor mediated currents, whereas coexpression of a non-functional but non-dominant negative P2X₄ mutant introduced P2X₄-like pharmacological properties to the whole cell current. When heterologously coexpressed, the extent of co-localization of P2X₄ and P2X₇ is low suggesting that the favoured assembly pathway is the formation of homotrimers. Native receptor currents with P2X_4/7-like properties have, however, been recorded from airway epithelial cells [45].

Although the electrophysiological properties of many homomeric and heteromeric receptors have been described, there are examples of P2X receptor currents recorded from native tissue with properties that do not match the recombinant receptors. These include ATP-evoked currents recorded from rat primary sensory neurons and from trigeminal mesencepthalic nucleus neurons [46], [47]. Either there are heteromeric assemblies that have not yet been characterized or other proteins interact with the receptors in native tissue to modify their properties.

Formation of multi-protein signaling complexes

P2X receptors functionally interact with other neighbouring P2X receptors and also with ion channels that belong to different structural classes including members of the Cys loop family and gap junction family. Underlying these interactions is a close spatial arrangement of the ion channel complexes within the plasma membrane. For P2X₂ receptors, the formation of stable clusters of homotrimers, as resolved by BN-PAGE analysis [17], correlates with the demonstration that the receptors within a patch of membrane open in a non-independent manner, displaying positive cooperativity [48–50]. P2X₂ receptors also interact with members of the Cys-loop family of ligand gated ion channels, including GABA_A, nicotinic and 5HT₃ receptors, all of which are involved in fast synaptic transmission [51–56]. In this case the effect of co-activation is cross-inhibition, as demonstrated initially in sympathetic neurones, where inward currents evoked by the simultaneous application of ATP and ACh were shown to be considerably smaller than the sum of the responses produced by individually applying the two agonists [57]. FRET analysis of heterologously coexpressed P2X₂ and nicotinic receptors indicates that they are located within 100 Å of each other suggesting that they form oligomers [58]. The mechanism underlying the reciprocol inhibitory cross-talk is poorly understood although there is evidence to suggest that it involves the C-terminal region of P2X₂ receptors [55], [59], [60]. This region is very divergent between the different subtypes and yet functional coupling with the Cys-loop channels is not restricted to P2X₂ receptors. It has also been shown for P2X₃ and GABA_A receptors in nociceptive DRG neurones and to require a QST motif within the C-terminus of P2X₃ [61]. The interaction between these two classes of receptors affects their trafficking as well as their function. The coexpression of P2X₂ with rho1/GABA receptors was shown to recruit the mainly intracellular rho1/GABA receptors to the plasma membrane suggesting that coassembly of these channels occurs within the ER such that they are targeted to synapses together [60].

P2X₇ receptors have a very different tissue distribution from P2X₂ and P2X₃ receptors, being predominantly expressed in immune and epithelial cells. They interact with another ion channel, pannexin-1 (panx1) [62] which belongs to a recently identified family of proteins (panx1-3) that share homology with invertebrate gap junction proteins innexins [63]. Despite this homology, recent evidence suggests that panx1 does not form intercellular gap junctions but instead functions as a hemichannel [64–66]. Panx1 is expressed in a variety of immune cells and also cell lines that have been used to characterize P2X₇ receptor function. P2X₇ receptor activation induces responses that are unique to this subtype, such as an increase in membrane permeability to large dyes and cytoskeletal rearrangements that result in plasma membrane blebbing. Knocking down panx1 expression was shown to inhibit P2X₇ receptor-mediated dye uptake but not membrane blebbing [62]. In immune cells, inhibiting panx1 reduces P2X₇ receptor stimulated release of IL-1β. The nature of the structural and functional association between P2X₇ and panx1 remains to be elucidated.

Ion channels are not the only proteins that are known to associate with P2X receptors. P2X₂ interacts with a neuronal scaffolding protein Fe65 and they co-localize at excitatory synapses in the CNS [67]. In addition a proline rich region within the C-terminus of P2X₂ binds βIII tubulin, suggesting that microtubules are involved in regulating this receptor [68]. A proteomic analysis of P2X₇-containing complexes purified from HEK293 cells identified 11 different proteins within the complex including β-actin, α-actinin, heat shock proteins and a receptor protein tyrosine phosphatase [69]. Many of these interactions are thought to involve the unique, long C-terminal region of P2X₇ but the functional significance of these interactions is not fully understood.

Trafficking of P2X receptors

The secretory pathway

P2X receptors are delivered to the plasma membrane along the secretory pathway via the ER and the Golgi complex. There are mechanisms along this pathway capable of either increasing or decreasing delivery of receptors to the plasma membrane and this is one way in which the responsiveness of the cell to extracellular ATP can be regulated. The efficiency with which P2X receptors assemble and traffic along the secretory pathway varies between the different subtypes and is also cell type-dependent.

As discussed earlier, P2X₆ receptors are retained in the ER as monomers but do traffic to the surface as heteromers with P2X₂ and P2X₄. P2X₅ is believed to have an ER retention motif which is at least partly responsible for its low functional expression as a homomer [70]. For P2X₃ receptors, increased trafficking along the secretory pathway appears to be an important mechanism for enhancing its activity in sensory neurones. Potentiation of P2X₃ receptor function is associated both with nerve injury induced allodynia and the algogenic action of calcitonin-gene-related peptide (CGRP) [71], [72]. In trigeminal neurones, pretreatment with CGRP was shown to stimulate trafficking of P2X₃ receptors to the cell surface via a brefeldin A sensitive pathway and this caused a strong and prolonged enhancement of receptor function that was dependent upon protein kinase (PK) A and PKC activation. The Ca^2 +/calmodulin dependent PK II has also been identified as being involved in regulating the trafficking of P2X₃ receptors; it becomes activated by increased electrical activity and promotes the surface expression of P2X₃ receptors in DRG neurones without changing their overall expression [73]. Confocal images showing the subcellular distribution of neuronal P2X₃ receptors are consistent with their redistribution from the ER to the cell surface, however the assembly state of the receptor has not been examined and so it is unknown whether the receptor is retained in monomeric or trimeric form.

For P2X₇ receptors, assembly and trafficking to the plasma membrane appears to be cell type dependent. In monocytes and lymphocytes the receptors are predominantly intracellular [74] whereas when monocytes differentiate into macrophages there is a large increase in their surface expression and function [75]. This change occurs without a significant increase in receptor mRNA levels, suggesting that the upregulation of surface P2X₇ involves a redistribution of intracellular receptors to the plasma membrane. In the brain, western blot analysis of P2X₇ receptors suggested they were predominantly in monomeric form and incompletely glycosylated, whereas in peritoneal macrophages, they were identified only as glycosylated multimeric complexes [76]. A later study from the same group, however, cast doubt on the specificity of the antibody when used for brain tissue [77]. Unlike P2X₆, homomeric P2X₇ receptors are functional and do not to require coassembly with other members of the P2X family in order to be exported from the ER. There are however several truncated splice variants of P2X₇ receptors, one of which was recently shown to assemble with the full-length variant and antagonize its functional expression [78], [79]. Another was shown to be expressed at high levels in the brain [79]. The expression of truncated splice variant could be a mechanism regulating the correct assembly and trafficking of P2X₇ receptors. In addition, P2X₇ receptors interact with many other proteins, including heat shock protein 90 (HSP90), which is a molecular chaperone able to prevent protein aggregation and to promote refolding of denatured proteins. This chaperone becomes tyrosine phosphorylated when associated with P2X₇, but whether or not it plays a role in assembly and targeting of the receptor is unknown [80]. The long C-terminus of the P2X₇ receptor is thought to be involved in many of the interactions with other proteins and within the distal C-terminus is a region with homology to lipopolysaccharide binding proteins [81]. Partial deletions and point mutations within this region, including a mutation linked to chronic lymphocytic leukemia [82], inhibit the plasma membrane expression of P2X₇ receptors and promote ER retention, whereas larger deletions restore both the expression and some of the functional properties of receptors at the plasma membrane [83], [84]. This suggests that neighbouring regions within the distal C-terminus are either directly or indirectly involved in regulating the exposure of a retention motif.

The endocytic pathway

For many ligand-gated ion channels, retrieval from and recyling to the plasma membrane is an important mechanism for rapidly modulating their function [85]. Alternatively receptors can be targeted to lysosomes for degradation leading to a reduced response to a ligand. The stabilization of P2X receptors at the plasma membrane requires a motif, YXXXK, present within the proximal C-terminal region of all P2X receptor subunits [31]. P2X₂ receptors are normally stably expressed at the cell surface, however, mutations at Y362 or K366, enhance their retrieval without affecting their trafficking through the secretory pathway. These mutations also disrupted the axonal targeting of P2X₂ receptors heterologously expressed in hippocampal neurones, suggesting that selective stabilization might be responsible for determining the location of the receptors. Despite the conservation of this motif throughout the P2X family, sustained exposure to agonist has been reported to cause the internalization of P2X₁, P2X₅ and P2X₇ receptors [86–91].

The role of endocytic pathways in regulating the surface expression of P2X receptors has been principally studied for the P2X₄ receptor. They undergo constitutive endocytosis, which proceeds rapidly via a dynamin and AP-2 dependent mechanism, and a tyrosine-based motif within the C-terminus and a dileucine-like motif within the N-terminus act independently to mediate this process and target the receptors to lysosomes [22], [92–94]. The tyrosine based motif takes the form of a novel YXXGL motif which can be recognized by AP-2 and binds to the same pocket on the µ2 subunit as YXXφ motifs. The endogenous P2X₄ receptors in immune and vascular endothelial cells are predominantly localized to lysosomes, and proteomic studies of lysosome resident membrane protein have identified this subtype but no other members of the family [95]. Similar to other lysosome-targeted proteins, P2X₄ resists degradation within this proteolytic environment by virtue of N-linked glycans and it is up-regulated at the plasma membrane following stimulation of lysosome exocytosis. The enhancement of P2X₄ receptor mediated currents indicates that lysosome resident receptors retain their functional integrity [94]. Why P2X₄ receptors are targeted to lysosomes is unclear. It is also unclear whether they always traffic via the plasma membrane or can take a direct route from the TGN to late endosomes as suggested for other proteins with a dileucine-targeting motif. Lysosomal targeting ensures low surface expression of the receptor in non-activated immune cells and this is likely to be important because an up-regulation of P2X₄ receptors in microglia following nerve injury contributes to neuropathic pain. Activation of P2X₇ receptors triggers lysosome exocytosis and this could potentially recruit P2X₄ receptors to the plasma membrane [96]. Lysosome exocytosis also occurs in response to membrane injury, when ATP will be released from the damaged cell which could lead to P2X₄ receptor activation [97], [99].

It is possible that P2X₄ receptors have a functional role within endo-lysosomes. Lysosomes have been shown to be physiologically important calcium stores [98] and the highly calcium permeable P2X₄ receptors may play a role in calcium flux between the lumen and the cytosol. P2X₄ receptors are also delivered to phagosomes and it is tempting to speculate they could form a sensor for ATP released from a phagocytosed bacterium or apoptotic cell [94]. A recent report on a P2X-like receptor from Dictyostelium was the first to show an intracellular role for these channels; these receptors are targeted to contractile vacuoles and are important for osmoregulation [99]. A mechanism for regulating the activity of intracellular P2X receptors has not been reported, although lysosomes are known to contain high levels of ATP [100].

Future perspectives

Studies into the assembly of P2X receptors have revealed surprising complexity in their interactions with each other, with structurally unrelated ion channels and with non-integral membrane proteins. Further analyses of these complexes with both low and high-resolution structural techniques will be able to more definitively address the composition of these heteromeric assemblies. Site directed mutagenesis combined with functional analyses should provide a means of uncoupling these interactions thereby revealing their molecular basis and functional importance.

Understanding the physiological roles of these assemblies is required because several of the receptors are important therapeutic targets and their coassembly is likely to alter their pharmacological properties, which may provide an opportunity for more specific drug design. Studies using mice with genes knocked out for multiple P2X subunits may further reveal the contribution of the heteromeric receptors towards ATP-mediated responses

The targeting of P2X receptors to their destinations within the cell also provides insight into their physiological roles. Elucidation of the mechanisms that control assembly and trafficking of the receptor complexes will provide a greater understanding of the importance of regulating plasma membrane in physiological and pathological processes.

Supplementary Material

Supplementary Table I. Physiological and pathological role of P2X receptors confirmed by knocking out or knocking down their expression.

	Distribution
P2X1	Smooth muscle	In P2X1-deficient mice there is a reduction of contraction of the vas deferens in response to sympathetic nerve stimulation, which results in reduced sperm in the ejaculate and loss of fertility [1].
		P2X1 receptors contribute to sympathetic-induced vasoconstriction in the smooth muscle of the mesenteric artery [2] and bladder [3].
P2X2	Central and peripheral nervous systems including sensory neurones	Mice deficient in P2X2 receptors show attenuated ventilatory response to hypoxia because of a reduction in the response of the carotid sinus nerve [4].
P2X3	Restricted to sensory neurones	Knockout of P2X3 receptors results in relatively small defects in inflammatory pain but obvious deficits in responsiveness to non-noxious stimuli [5]. The use of antisense oligonucleotides, however, showed a more significant role for P2X3 in chronic neuropathic and inflammatory pain [6]. Knockout of P2X3 and P2X2 also caused urinary bladder hyporeflexia [7], reduced afferent nerve activity in response to bladder distension [8] impaired peristalsis [9] and elimination of taste responses in gustatory nerve [10].
P2X4	Widespread throughout peripheral tissues, including immune, epithelial, endothelial and cardiac tissue and the CNS.	Using antisense oligonucleotides, receptors were shown to mediate a calcium influx into vascular endothelial cells in response to shear stress, which triggers secretion of nitric oxide [11], [12]. P2X4 knockout mice show impaired flow induced release of NO and defects in both the control of blood pressure and vascular remodelling [13].
		In human airway epithelial cells, knockdown of P2X4 and P2X6 by siRNA inhibited zinc-induced calcium entry [14]. This calcium influx was shown to restore chloride secretion across cystic fibrosis airway epithelia [15], suggesting that these receptors are possible therapeutic targets for treatment of cystic fibrosis [16].
		Receptors in spinal cord microglia/macrophages play an important role in peripheral nerve injury-induced pain hypersensitivity (tactile allodynia) [17]. P2X4 receptor expression is increased in spinal cord microglia following nerve injury and antisense oligonucleotides against the P2X4 receptor reduced the induction of tactile allodynia [17].
		In the CNS, long term potentiation at hippocampal synapses was reduced in P2X4 knock out mice suggesting a role for calcium entry through these receptors contributing to synaptic plasticity [18].
P2X7	Immune cells, epithelial cells.	Knockdown of the P2X7 receptor abolished the ATP-stimulated processing and release of Il-1β in mouse macrophages [19]. These mice also show reduced cartilage destruction in an antibody-induced arthritis state [20] and reduced bone formation [21].
		Disruption of the P2X7 gene reduced inflammatory and neuropathic pain [22].
		Loss-of-function polymorphisms in P2X7 receptors are linked to epithelial cell cancers and lymphomas [23], [24].

Acknowledgements

We would like to thank Drs J. M. Edwardson and I. Greger for providing helpful comments on this manuscript. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.