PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of transbThe Royal Society PublishingPhilosophical Transactions BAboutBrowse By SubjectAlertsFree Trial
 
Philos Trans R Soc Lond B Biol Sci. 2016 August 5; 371(1700): 20150427.
PMCID: PMC4938027

P2X receptors

Abstract

Extracellular adenosine 5′-triphosphate (ATP) activates cell surface P2X and P2Y receptors. P2X receptors are membrane ion channels preferably permeable to sodium, potassium and calcium that open within milliseconds of the binding of ATP. In molecular architecture, they form a unique structural family. The receptor is a trimer, the binding of ATP between subunits causes them to flex together within the ectodomain and separate in the membrane-spanning region so as to open a central channel. P2X receptors have a widespread tissue distribution. On some smooth muscle cells, P2X receptors mediate the fast excitatory junction potential that leads to depolarization and contraction. In the central nervous system, activation of P2X receptors allows calcium to enter neurons and this can evoke slower neuromodulatory responses such as the trafficking of receptors for the neurotransmitter glutamate. In primary afferent nerves, P2X receptors are critical for the initiation of action potentials when they respond to ATP released from sensory cells such as taste buds, chemoreceptors or urothelium. In immune cells, activation of P2X receptors triggers the release of pro-inflammatory cytokines such as interleukin 1β. The development of selective blockers of different P2X receptors has led to clinical trials of their effectiveness in the management of cough, pain, inflammation and certain neurodegenerative diseases.

This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’.

Keywords: adenosine 5′-triphosphate, ion channel, purinergic signalling

1. Beginnings of the field

The study of the proteins that came to be known as P2X receptors has three distinct origins. The earliest was the description by Burnstock & Holman [1,2] of the junction potentials (ejps) recorded with glass microelectrodes from smooth muscle cells of the guinea pig vas deferens. By analogy with the similar depolarizations observed in skeletal muscle (endplate potentials), the inference was that the transmitter released from the nerve briefly increased the permeability of the smooth muscle membrane to cations. Noradrenalin was thought to be the transmitter released from the sympathetic nerves, a conclusion buttressed by the finding in 1970 that pretreatment with 6-hydroxydopamine to destroy sympathetic nerves abolished the ejp [3].

By 1978, evidence was becoming compelling that adenosine 5′-triphosphate (ATP) was the sympathetic transmitter in the bladder [4] and the use of the analogue αβ-methylene-ATP (αβmeATP) as a desensitizing blocker extended this conclusion to the vas deferens [5]. In 1985, Burnstock & Kennedy [6] characterized such actions of ATP, which were mimicked and/or blocked by αβmeATP, as involving P2X receptors. These were distinguished from P2Y receptors, at which 2-methyl-thio-ATP was a more effective agonist and activation of which typically led to smooth muscle relaxation. It is important to recognize that these studies on the vas deferens and bladder had an intrinsically physiological context, in the sense that they were directed at understanding the effects of ATP released from nerve cells, specifically post-ganglionic sympathetic nerves.

The other two origins of P2X receptors were more pharmacological, in the sense that they involved studies of the action of exogenous ATP. One was the observation that ATP caused the release of histamine from mast cells and that this was associated with an increase in permeability of the mast cell membrane [7]. The most effective form of ATP appeared to be ATP4−. This tetrabasic form of the molecule forms only a small fraction of the total ATP in physiological solutions, most being complexed as MgATP or CaATP. The permeability increase was monitored as the release of 32P-labelled intermediary metabolites such as phosphatidyl inositol following pre-loading of the cells with 32P inorganic phosphate, but it could also be followed as the uptake of fluorescent dyes such as ethidium or propidium [8]. Several other cells types were found that responded to ATP4−, including macrophages, neutrophils, gland cells and endothelium, and the receptor involved became called P2Z (reviewed in [9]).

The third original approach was electrophysiological: in 1983, three separate groups studied the action of exogenous ATP on membrane potential or currents. Recording from sensory neurons cultured from rat dorsal root ganglia, Krishtal and co-workers [10] showed that ATP (1–100 µM) evoked an inward membrane current within milliseconds of its application. This resulted from an increase in conductance to cations. Kolb & Wakelam [11] recorded the single channel currents elicited by application of ATP (10 µM) to chicken muscle cells, which had properties indicating an increase in conductance to sodium and potassium ions. Later that year, Jahr & Jessell [12] confirmed the findings of Krishtal et al. and further distinguished the rapidly desensitizing depolarization of rat dorsal root ganglion cells induced by ATP from the more sustained depolarization observed in neurons cultured from the dorsal horn of the spinal cord.

More detailed biophysical characterization of ATP-induced currents followed, for smooth muscle [13], sensory neurons [14,15], pheochromocytoma cells [16] and locus coeruleus neurons [17], and three papers presented additional evidence for synaptic transmission mediated directly by ATP [1820]. On the other hand, the lack of selective and potent antagonists hampered any conclusive demonstration of the physiological function of ATP-operated P2X receptors.

2. Following cDNA cloning

The decade beginning in 1983 was a period in which cDNAs were isolated for almost all membrane ion channels [21,22] and P2X receptors joined this group in 1994. A team at the Glaxo Institute for Molecular Biology in Geneva [23] identified a cDNA encoding the P2X1 receptor by injecting oocytes with progressive fractions of a cDNA expression library made from RNA extracted from the rat vas deferens. A similar approach starting with the RNA from PC12 cells was used by Brake et al. [24] to clone the P2X2 receptor. The five further members of the family were identified by homology-based approaches from a wide range of tissues [25] and the seven mammalian genes were subsequently identified. The properties of the P2X7 receptor, most notably its relative low sensitivity to ATP and the permeability to larger molecular weight dyes, indicated that it corresponded to the P2Z receptor named by Gordon in 1986 [9]. It was found that P2X receptor genes were widely expressed throughout vertebrates and lower eukaryotic organisms, but unlike several other ion channels they have not been found in prokaryotes. The genes can be identified in green algae, which represent the earliest separation of animals from plants ([26], see also [27]). In the slime mould Dictyostelium discoides, P2X receptors have a predominantly intracellular distribution and function [28,29].

Many inferences about molecular structure became possible as a result of the expression of cDNAs, particularly when biophysical measurements were combined with mutagenesis. It was immediately clear that each P2X receptor subunit had two membrane-spanning domains (TM1 and TM2), with intracellular N- and C-terminus, and that most of the protein was located as a large ectodomain. Several lines of evidence indicated that the functional protein was a trimer: indeed, three ATP-binding sites had been suggested by Bean [14] on the basis of his ATP dose–response curves from bullfrog sensory neurons. This evidence included biochemical approaches using blue native polyacrylamide gel electrophoresis [30] and functional approaches with co-expression and concatenation of subunits carrying reporter mutations [3135]. The demonstration that P2X receptors were trimers set them in clear distinction from the tetrameric glutamate-gated ion channels and the pentameric nicotinic superfamily (which also includes channels gated by glycine, γ-aminobutryic acid and 5-hydroxytryptamine; [22]). These approaches also showed that functional channels could form as hetero- or homo-trimers (e.g. P2X2/3 and P2X1/5 receptors; [25]).

The P2X receptor genes contain 10–12 introns and are found on five chromosomes [25]. The genes for P2X1 and PX5 receptors, and for P2X4 and P2X7 receptors, are adjacent: this presumably reflects relatively recent duplication. The only channelopathy described is a loss-of-function mutation in the P2X2 receptor that results in hearing loss in Chinese [36] and Italian [37] families. Other efforts to associate single nucleotide polymorphisms with disease propensity in humans have revealed several but rather weak associations: these have been comprehensively reviewed for the P2X7 receptor [37,38].

Determination of the distribution of the P2X receptors also followed the molecular cloning, at either the RNA or protein level ([3942], see also [25]). These studies indicated that P2X receptors were much more widely expressed through vertebrate tissues than had been previously anticipated on the basis of functional studies. Notable examples were the predominant expression of P2X2, P2X4 and P2X6 subunits in the central nervous system, the abundance of P2X4 and P2X7 receptors in glandular tissue and immune cells and the very limited distribution of P2X3 subunits in a subset of sensory neurons involved in taste, bladder filling, baroreception and certain modalities of pain [25,43].

It has become clear that ATP does not have any widespread direct role as a fast neurotransmitter in the central nervous system [43,44]. P2X receptors are found on glia and neurons. On astrocytes in mouse cortex, the predominant form is a P2X1/P2X5 heteromer [45], whereas microglia express mostly the P2X7 receptor [40,46]. On neurons in the hippocampus, P2X4 receptors are located at the periphery of the post-synaptic density [47]. Recent evidence suggests that ATP released from astrocytes can activate these receptors and lead to a reduction in the trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors to the synapse [48]. The P2X4 receptor has a relatively high calcium permeability [49] which, unlike the case of the NMDA receptor, allows calcium entry at both hyperpolarized and depolarized membrane potentials. This calcium entry appears to inhibit AMPA receptor trafficking by a mechanism involving calmodulin-dependent kinase II and/or a calcium-dependent phosphatase [48]. A wider role for P2X receptors in the operation or trafficking of other ion channels has long been suspected [5052] and is now beginning to be worked out in molecular detail [5355].

In the peripheral nervous system, important new roles for P2X receptors in afferent signalling have been established. ATP is released from chemosensing cells to activate P2X receptors in terminals of the carotid sinus nerve [56]. ATP release by taste buds is the first step in all modalities of taste sensation: the ATP activates P2X2/P2X3-subunit-containing receptors on the gustatory nerve [57]. An analogous signalling role from urothelium to primary afferent nerves has been inferred for sensing bladder distension [58].

Release of inflammatory cytokines by ATP was well known before the cDNA cloning, but the expression of P2X7 receptors on a wide range of immune cells has prompted an intensive study of their role in inflammation [59,60]. The availability of mice lacking P2X7 receptors, as well as the development of a range of antagonists, is now documenting roles for ATP signalling in skin [61], bone [62,63], glandular epithelium [64] and cancer cells [65].

3. Molecular modus operandi

The determination of the structure of a truncated zebrafish P2X4 receptor by X-ray crystallography (closed state [66]; open state and closed states [67]) essentially confirmed the inferences based on 15 years of work of mutagenesis combined with functional expression [68,69]. Of course, it achieved much more than that. It provided an immediate structural view of a novel family of membrane proteins, in which the pore-forming region extends to include the epithelial sodium channel (ENaC) and acid-sensing ion channel (ASIC) [70]. It indicated a new type of gated pore formation in biological membranes. It explained how ATP was a selective agonist without the requirement for hydrolysis [71]. It provided the atomic template for drug development (see reviews [68,72,73]).

Each protomer of the truncated P2X receptor resembles a dolphin [66]. The tail flukes are missing but would be positioned inside the cell, with the posterior part of the body (the peduncle) traversing the membrane, and the bulk of the body with dorsal fin, left and right flippers, head and beak protruding into the extracellular solution. The contacts with ATP are provided by eleven amino acids, four from one subunit and seven from another [67] (figure 1). The binding of ATP results in the head domain of one dolphin subunit being pulled downwards toward the left flipper of the adjacent subunit (figure 1). This torsions the body region outwards so as to increase the separation between the three subunits, opening three lateral portals through which ions can enter the central vestibule. It also pulls apart the six transmembrane domains, of which the three TM2 line the central axis of the permeation pathway (figure 1).

Figure 1.
P2X receptor structure, the ATP-binding site and the permeation pathway. (a) Assembly of P2X2 receptors from three subunits, depicted in closed (left) and open (right) states. Upper, middle and lower panels show one, two and three subunits, respectively. ...

These movements can be readily deduced by a comparison of the crystal structures of the closed [66] and open (ATP-bound) [67] states, and they are strongly supported by several functional approaches. For example, disulfide locking of pairs of engineered cysteine residues has provided direct evidence for the approximation of amino acid residues [32,7784]. The open–closed transition of the receptor can also be driven by light in the complete absence of ATP, when a light-sensitive azobenzene molecule is incorporated into the receptor by attachment to cysteine residues in two different subunits [85,86]. The cis–trans isomerization of the azobenzene by 440 nm irradiation pushes apart residues (P329C) in two adjacent subunits to open the channel, and the trans–cis conformational change at 360 nm conversely closes the channel [86]. A third application of cysteine substitution has been made by attaching lipophilic side chains at a position likely to move through the outer lipid leaflet of the membrane. Thus, in the P2X2[I328C] receptor, the addition of a propyl-methanethiosulfonate is as effective to open the channel as ATP itself [87].

From the structural point of view, the zebrafish P2X4 receptor that was used for X-ray crystallography lacks both the intracellular N- and C-terminus. Although such truncated molecules do form functional channels [66], it is known that the parts of these termini that are close to the transmembrane domains contain highly conserved short amino acid sequences that are important determinants of normal expression and function. These are YXTX[K/R] before TM1, and YXXXK after TM2. It is not difficult to imagine the folding of six YXXXK domains (two from each subunit) so as to form a stable ‘hanging basket’ in the cytoplasm internal to the inner opening of the permeation pathway, stabilized perhaps by interactions between the positive lysine side chains and the π electron clouds of the Tyr residues. Currents evoked by ATP at P2X1 receptors show rapid desensitization (within tens of milliseconds), whereas currents at P2X2 receptors are sustained for several seconds. Transfer of the protein segments immediately before TM1 has profound effects on this desensitization [88]. Similarly, deletion of a cysteine-rich region that is found after TM2 only in the P2X7 receptor has effects on the time course of the ATP-induced current as well as on the passage of large fluorescent dyes through the P2X7 channel [89]. There will be much more to learn from a crystal structure of the holoprotein.

The present open channel structure provides a pathway through the membrane for small cations. In the zebrafish P2X4 receptor the Cα atoms of the alanine (A347) residues lie on a circle with diameter of 1.2 nm [67], and the same is true for S342, which lies at the narrowest part of the pore in models of the rat P2X7 receptor [90]. However, the activated rat P2X7 receptor allows the permeation of dyes as large as sulforhodamine methane thiosulfonate, which has dimensions of 0.90 × 1.40 × 1.65 nm [90]. This implies that some P2X receptors can also adopt conformations with a wider permeation pathway. The ASIC also forms a permeation pathway by obliquely intersecting TM2 segments. It has been shown to exhibit two open states, one with a wider permeation pathway, depending on the extracellular pH [91].

4. Therapeutic exploitation

P2X7 receptors have been the most intensively investigated and many pharmaceutical companies have synthesized small molecules that are potent and selective blockers of the human receptor [92]. These include Abbott, Actelion, Affectis, AstraZeneca, Evotec, GlaxoSmithKline, Janssen, Johnson and Johnson, Merck, Neurogen, Nissan Chemical, Pfizer, Roche and Schering [92,93]. Because the activation of P2X7 receptors by ATP is a key step in the release of inflammatory cytokines from microglia primed with bacterial lipopolysaccharide [94], P2X receptors have long been considered as possible therapeutic targets in inflammatory pain [95,96]. Initial hopes that they may have efficacy were disappointed in Phase IIb trials in rheumatoid arthritis [97,98]. Ongoing studies include preclinical work using animal models of neuropsychiatric and neurodegenerative disease [38].

P2X3 receptors have a very limited distribution on primary afferent fibres and they have been targeted for visceral pain [99] and, more recently, for the treatment of chronic cough. A P2X3 receptor antagonist from Afferent Pharmaceuticals (AF-219) was an effective antitussive in a randomized, double-blind, placebo-controlled phase 2 study [100,101]. One confounding factor with such trials of P2X3 receptor antagonists will be the difficulty in conducting ‘blind’ trials. ATP is a transmitter released by taste buds and it activates P2X3-subunit-containing receptors on gustatory nerves [57], and all the patients in the trial who took AF-219 reported disturbance of taste [99].

P2X1 receptors were first identified in the vas deferens and mice lacking the receptor have no ejps and impaired ejaculation [102]. Noradrenalin that is also released from sympathetic nerves contracts the vas deferens by activating α1A adrenoceptors, but without membrane depolarization. The possibility of further exploiting these effects in the development of a male contraceptive has recently been boosted by the demonstration that mice lacking both P2X1 receptors and α1A receptors are completely infertile [103].

Acknowledgement

I thank all my former students, colleagues, collaborators and competitors, and particularly Liam Browne for help with molecular modelling.

Competing interests

I declare I do not have any competing interest.

Funding

Work in my laboratory was supported by the Wellcome Trust.

References

1. Burnstock G, Holman ME 1960. Autonomic nerve–smooth muscle transmission. Nature 187, 951–952. (doi:10.1038/187951a0) [PubMed]
2. Burnstock G, Holman ME 1961. The transmission of excitation from autonomic nerve to smooth muscle. J. Physiol. 155, 115–133. (doi:10.1113/jphysiol.1961.sp006617) [PubMed]
3. Furness JB, Campbell GR, Gillard SM, Malmfors T, Cobb JLS, Burnstock G 1970. Cellular studies of sympathetic denervation produced by 6-hydroxydopamine in the vas deferens. J. Pharmacol. Exp. Ther. 174, 111–122. [PubMed]
4. Burnstock G, Cocks T, Crowe R, Kasakov L 1978. Purinergic innervation of the guinea pig urinary bladder. Br. J. Pharmacol. 63, 125–138. (doi:10.1111/j.1476-5381.1978.tb07782.x) [PMC free article] [PubMed]
5. Sneddon P, Burnstock G 1984. Inhibition of excitatory junction potentials in guinea pig vas deferens by alpha,beta-methylene-ATP: further evidence for ATP and noradrenaline as cotransmitters. Eur. J. Pharmacol. 100, 85–90. (doi:10.1016/0014-2999(84)90318-2) [PubMed]
6. Burnstock G, Kennedy C 1985. Is there a basis for distinguishing two types of P2-purinoceptor? Gen. Pharmacol. 16, 433–440. (doi:10.1016/0306-3623(85)90001-1) [PubMed]
7. Cockcroft S, Gomperts BD 1979. ATP induces nucleotide permeability in rat mast cells. Nature 279, 541–542. (doi:10.1038/279541a0) [PubMed]
8. Tatham PE, Cusack NJ, Gomperts BD 1988. Characterisation of the ATP4− receptor that mediates permeabilisation of rat mast cells. Eur. J. Pharmacol. 147, 13–21. (doi:10.1016/0014-2999(88)90628-0) [PubMed]
9. Gordon JL. 1986. Extracellular ATP: effects, sources and fates. Biochem. J. 233, 309–319. (doi:10.1042/bj2330309) [PubMed]
10. Krishtal OA, Marchenko SM, Pidoplichko VI 1983. Receptor for ATP in the membrane of mammalian sensory neurons. Neurosci. Lett. 35, 41–45. (doi:10.1016/0304-3940(83)90524-4) [PubMed]
11. Kolb HA, Wakelam MJO 1983. Transmitter-like action of ATP on patched membranes of cultured myoblasts and myotubes. Nature 303, 621–623. (doi:10.1038/303621a0) [PubMed]
12. Jahr CE, Jessell TM 1983. ATP excites a subpopulation of rat dorsal horn neurons. Nature 304, 730–733. (doi:10.1038/304730a0) [PubMed]
13. Benham CD. 1989. ATP-activated channels gate calcium entry in single smooth muscle cells dissociated from rabbit ear artery. J. Physiol. 419, 689–701. (doi:10.1113/jphysiol.1989.sp017893) [PubMed]
14. Bean BP. 1990. ATP-activated channel in rat and bullfrog sensory neurons: concentration dependence and kinetics. J. Neurosci. 10, 1–10. [PubMed]
15. Bean BP, Williams CA, Cellen PW 1990. ATP-activated channel in rat and bullfrog sensory neurons: current–voltage relation and single channel behavior. J. Neurosci. 10, 11–19. [PubMed]
16. Nakazawa K, Fujimori K, Takanaka A, Inoue K 1991. Comparison of adenosine triphosphate- and nicotine-activated inward currents in rat phaeochromocytoma cells. J. Physiol. 434, 647–660. (doi:10.1113/jphysiol.1991.sp018491) [PubMed]
17. Shen K-Z, North RA 1993. Excitation of rat locus coeruleus by adenosine 5′- triphosphate: ionic mechanism and receptor characterization. J. Neurosci. 13, 894–899. [PubMed]
18. Evans RJ, Derkach V, Surprenant A 1992. ATP mediates fast synaptic transmission in mammalian neurons. Nature 357, 503–505. (doi:10.1038/357503a0) [PubMed]
19. Edwards FA, Gibb AJ, Colquhoun D 1992. ATP receptor-mediated synaptic current in the central nervous system. Nature 359, 144–146. (doi:10.1038/359144a0) [PubMed]
20. Silinsky EM, Gerzanich V, Vanner SM 1992. ATP mediates excitatory synaptic transmission in mammalian neurons. Br. J. Pharmacol. 106, 762–763. (doi:10.1111/j.1476-5381.1992.tb14408.x) [PMC free article] [PubMed]
21. North RA. (ed.). 1995. Ligand- and voltage-gated ion channels. Boca Raton, FL: CRC Press.
22. Hille B. 2001. Ion channels of excitable membranes, 3rd edn Sunderland, MA: Sinauer.
23. Valera S, Hussy N, Evans RJ, Adami N, North RA, Surprenant A, Buell G 1994. A new class of ligand-gated ion channel defined by P2X receptor for extracellular ATP. Nature 371, 516–519. (doi:10.1038/371516a0) [PubMed]
24. Brake AJ, Wagenbach MJ, Julius D 1994. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature 371, 519–524. (doi:10.1038/371519a0) [PubMed]
25. North RA. 2002. Molecular physiology of P2X receptors. Physiol. Rev. 82, 1013–1067. (doi:10.1152/physrev.00015.2002) [PubMed]
26. Fountain SJ, Cao L, Young MT, North RA 2008. Permeation properties of a P2X receptor in the green algae Ostreococcus tauri. J. Biol. Chem. 283, 15 122–15 126. (doi:10.1074/jbc.M801512200) [PMC free article] [PubMed]
27. Surprenant A, North RA 2009. Signalling at purinergic P2X receptors. Annu. Rev. Physiol. 71, 333–359. (doi:10.1146/annurev.physiol.70.113006.100630) [PubMed]
28. Fountain SJ, Parkinson K, Young MT, Cao L, Thompson CR, North RA 2007. An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum. Nature 448, 200–203. (doi:10.1038/nature05926) [PMC free article] [PubMed]
29. Parkinson K, Baines AE, Keller T, Gruenheit N, Bragg L, North RA, Thompson CR 2014. Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel. Nat. Cell. Biol. 16, 87–98. (doi:10.1038/ncb2887) [PMC free article] [PubMed]
30. Nicke A, Baumert HG, Rettinger J, Eichele A, Lambrecht G, Mutschler E, Schmalzing G 1998. P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO J. 17, 3016–3028. (doi:10.1093/emboj/17.11.3016) [PubMed]
31. Stoop R, Thomas S, Rassendren F, Kawashima E, Buell G, Surprenant A, North RA 1999. Contribution of individual subunits to the multimeric P2X2 receptor: estimates based on methanethiosulphonate block at T336C. Mol. Pharmacol. 56, 973–981. [PubMed]
32. Jiang LH, Kim M, Spelta V, Bo X, Surprenant A, North RA 2003. Subunit arrangement in P2X receptors. J. Neurosci. 23, 8903–8910. [PubMed]
33. Wilkinson WA, North RA 2006. Role of ectodomain lysines in the subunits of heteromeric P2X2/3 receptor. Mol. Pharmacol. 70, 1159–1163. (doi:10.1124/mol.106.026658) [PubMed]
34. Browne LE, Cao L, Broomhead HE, Bragg L, Wilkinson WJ, North RA 2011. P2X receptor channels show three-fold symmetry in ionic charge selectivity and unitary conductance. Nat. Neurosci. 14, 17–18. (doi:10.1038/nn.2705) [PMC free article] [PubMed]
35. Stelmashenko O, Lalo U, Yang Y, Bragg L, North RA, Compan V 2012. Activation of trimeric P2X2 receptors by fewer than three ATP molecules. Mol. Pharmacol. 82, 760–766. (doi:10.1124/mol.112.080903) [PubMed]
36. Yan D, et al. 2013. Mutation of the ATP-gated P2X(2) receptor leads to progressive hearing loss and increased susceptibility to noise. Proc. Natl Acad. Sci. USA 110, 2228–2233. (doi:10.1073/pnas.1222285110) [PubMed]
37. Faletra F, Girotto G, D'Adamo AP, Vozzi D, Morgan A, Gasparini P 2014. A novel P2RX2 mutation in an Italian family affected by autosomal dominant nonsyndromic hearing loss. Gene 534, 236–239. (doi:10.1016/j.gene.2013.10.052) [PubMed]
38. Bartlett R, Stokes L, Sluyter R 2014. The P2X7 receptor channel: recent developments and the use of P2X7 antagonists in models of disease. Pharmacol. Rev. 66, 638–675. (doi:10.1124/pr.113.008003) [PubMed]
39. Collo G, North RA, Kawashima E, Merlo-Pich E, Neidhart S, Surprenant A, Buell G 1996. Cloning of P2X5 and P2X6 receptors, and the distribution and properties of an extended family of ATP-gated ion channels. J. Neurosci. 16, 2495–2507. [PubMed]
40. Collo G, Neidhart S, Kawashima E, Kosco-Vilbois M, North RA, Buell G 1997. Tissue distribution of the P2X7 receptor. Neuropharmacology 36, 1277–1284. (doi:10.1016/S0028-3908(97)00140-8) [PubMed]
41. Vulchanova L, Riedl MS, Shuster SJ, Buell G, Surprenant A, North RA, Elde RP 1997. Immunohistochemical study of the P2X2 and P2X3 receptor subunits in monkey and rat sensory neurons and their central terminals. Neuropharmacology 36, 1229–1242. (doi:10.1016/S0028-3908(97)00126-3) [PubMed]
42. Nörenberg W, Illes P 2000. Neuronal P2X receptors: localisation and functional properties. Naunyn Schmiedebergs Arch. Pharmacol. 362, 324–339. (doi:10.1007/s002100000311) [PubMed]
43. Khakh BS, North RA 2012. Neuromodulation by extracellular ATP and P2X receptors in the CNS. Neuron 76, 51–69. (doi:10.1016/j.neuron.2012.09.024) [PMC free article] [PubMed]
44. Baxter AW, Choi SJ, Sim JA, North RA 2011. Role of P2X4 receptors in synaptic strengthening in mouse CA1 hippocampal neurons. Eur. J. Neurosci. 34, 213–220. (doi:10.1111/j.1460-9568.2011.07763.x) [PMC free article] [PubMed]
45. Lalo U, Pankratov Y, Wichert SP, Rossner MJ, North RA, Kirchhoff F, Verkhratsky A 2008. P2X1 and P2X5 subunits form the functional P2X receptor in mouse cortical astrocytes. J. Neurosci. 28, 5473–5480. (doi:10.1523/JNEUROSCI.1149-08.2008) [PMC free article] [PubMed]
46. Sim JA, Young MT, Sung HY, North RA, Surprenant A 2004. Reanalysis of P2X7 receptor expression in rodent brain. J. Neurosci. 24, 6307–6314. (doi:10.1523/JNEUROSCI.1469-04.2004) [PubMed]
47. Rubio ME, Soto F 2001. Distinct localization of P2X receptors at excitatory postsynaptic specializations. J. Neurosci. 21, 641–653. [PubMed]
48. Pougnet JT, Toulme E, Martinez A, Choquet D, Hosy E, Boué-Grabot E 2014. ATP P2X receptors down-regulate AMPA receptor trafficking and postsynaptic efficacy in hippocampal neurons. Neuron 83, 417–430. (doi:10.1016/j.neuron.2014.06.005) [PubMed]
49. Egan TM, Khakh BS 2004. Contribution of calcium ions to P2X channel responses. J. Neurosci. 24, 3413–3420. (doi:10.1523/JNEUROSCI.5429-03.2004) [PubMed]
50. Searl TJ, Redman RS, Silinsky EM 1998. Mutual occlusion of P2X ATP receptors and nicotinic receptors on sympathetic neurons of the guinea-pig. J. Physiol. 510, 783–791. (doi:10.1111/j.1469-7793.1998.783bj.x) [PubMed]
51. Barajas-López C, Espinosa-Luna R, Zhu Y 1998. Functional interactions between nicotinic and P2X channels in short-term cultures of guinea-pig submucosal neurons. J. Physiol. 513, 671–683. (doi:10.1111/j.1469-7793.1998.671ba.x) [PubMed]
52. Zhou X, Galligan JJ 1998. Non-additive interaction between nicotinic cholinergic and P2X purine receptors in guinea-pig enteric neurons in culture. J. Physiol. 513, 685–697. (doi:10.1111/j.1469-7793.1998.685ba.x) [PubMed]
53. Rodriguez RJ, et al. 2016. Presynaptic P2X1-3 and α3-containing nicotinic receptors assemble into functionally interacting ion channels in the rat hippocampus. Neuropharmacology 105, 241–257. (doi:10.1016/j.neuropharm.2016.01.022) [PubMed]
54. Limapichat W, Dougherty DA, Lester HA 2014. Subtype-specific mechanisms for functional interaction between α6β4 nicotinic acetylcholine receptors and P2X receptors. Mol. Pharmacol. 86, 263–274. (doi:10.1124/mol.114.093179) [PubMed]
55. Emerit MB, Baranowski C, Diaz J, Martinez A, Areias J, Alterio J, Masson J, Boué-Grabot E, Darmon M 2016. A new mechanism of receptor targeting by interaction between two classes of ligand-gated ion channels. J. Neurosci. 36, 1456–1470. (doi:10.1523/JNEUROSCI.2390-15.2016) [PubMed]
56. Piskuric NA, Nurse CA 2013. Expanding role of ATP as a versatile messenger at carotid and aortic body chemoreceptors. J. Physiol. 591, 415–422. (doi:10.1113/jphysiol.2012.234377) [PubMed]
57. Vandenbeuch A, Larson ED, Anderson CB, Smith SA, Ford AP, Finger TE, Kinnamon SC 2015. Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice. J. Physiol. 593, 1113–1125. (doi:10.1113/jphysiol.2014.281014) [PubMed]
58. Burnstock G. 2014. Purinergic signaling in the urinary tract in health and disease. Purinergic Signall. 10, 103–155. (doi:10.1007/s11302-013-9395-y) [PMC free article] [PubMed]
59. Di Virgilio F. 2015. Purinergic signaling in the immune system. Curr. Med. Chem. 22, 866–877. (doi:10.2174/0929867322666141210155311) [PubMed]
60. Di Virgilio F, Vuerich M 2015. P2X receptors and inflammation. Auton. Neurosci. 191, 117–123. (doi:10.1016/j.autneu.2015.04.011) [PubMed]
61. da Silva GL, Sperotto ND, Borges TJ, Bonorino C, Takyia CM, Coutinho-Silva R, Campos MM, Zanin RF, Morrone FB 2013. P2X7 receptor is required for neutrophil accumulation in a mouse model of irritant contact dermatitis. Exp. Dermatol. 22, 184–188. (doi:10.1111/exd.12094) [PubMed]
62. Jørgensen NR, Syberg S, Ellegaard M 2015. The role of P2X receptors in bone biology. Curr. Med. Chem. 22, 902–914. (doi:10.2174/0929867321666141215094749) [PubMed]
63. Agrawal A, Gartland A 2015. P2X7 receptors: role in bone cell formation and function. J. Mol. Endocrinol. 54, R75–R88. (doi:10.1530/JME-14-0226) [PubMed]
64. Woods LT, Camden JM, Batek JM, Petris MJ, Erb L, Weisman GA 2012. P2X7 receptor activation induces inflammatory responses in salivary gland epithelium. Am. J. Physiol. Cell Physiol. 303, C790–C801. (doi:10.1152/ajpcell.00072.2012) [PubMed]
65. Giannuzzo A, Pedersen SF, Novak I 2015. The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells. Mol. Cancer 14, 203–213. (doi:10.1186/s12943-015-0472-4) [PMC free article] [PubMed]
66. Kawate T, Michel JC, Birdsong WT, Gouaux E 2009. Crystal structure of the ATP-gated P2X4 ion channel in the closed state. Nature 460, 592–598. (doi:10.1038/nature08198) [PMC free article] [PubMed]
67. Hattori M, Gouaux E 2012. Molecular mechanism of ATP binding and ion channel activation in P2X receptors. Nature 485, 207–212. (doi:10.1038/nature11010) [PMC free article] [PubMed]
68. Browne LE, Jiang LH, North RA 2010. New structure enlivens interest in P2X receptors. Trends Pharmacol. Sci. 31, 229–237. (doi:10.1016/j.tips.2010.02.004) [PMC free article] [PubMed]
69. Evans RJ. 2010. Structural interpretation of P2X receptor mutagenesis studies on drug action. Br. J. Pharmacol. 161, 961–971. (doi:10.1111/j.1476-5381.2010.00728.x) [PMC free article] [PubMed]
70. Baconguis I, Hattori M, Gouaux E 2013. Unanticipated parallels in architecture and mechanism between ATP-gated P2X receptors and acid sensing ion channels. Curr. Opin. Struct. Biol. 23, 277–284. (doi:10.1016/j.sbi.2013.04.005) [PMC free article] [PubMed]
71. Hausmann R, Kless A, Schmalzing G 2015. Key sites for P2X receptor function and multimerization: overview of mutagenesis studies on a structural basis. Curr. Med. Chem. 22, 799–818. (doi:10.2174/0929867322666141128163215) [PMC free article] [PubMed]
72. Jiang R, Taly A, Grutter T 2013. Moving through the gate in ATP-activated receptors. Trends Biochem. 38, 20–29. (doi:10.1016/j.tibs.2012.10.006) [PubMed]
73. Habermacher C, Dunning K, Chataigneau T, Grutter T 2015. Molecular structure and function of P2X receptors. Neuropharmacology 104, 18–30. (doi:10.1016/j.neuropharm.2015.07.032) [PubMed]
74. Sali A, Blundell TL 1993. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815. (doi:10.1006/jmbi.1993.1626) [PubMed]
75. Davis IW. 2007. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383. (doi:10.1093/nar/gkm216) [PMC free article] [PubMed]
76. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE 2004. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612. (doi:10.1002/jcc.20084) [PubMed]
77. Marquez-Klapa B, Rettinger J, Bhargava Y, Eisele T, Nicke A 2007. Identification of an intersubunit cross-link between substituted cysteine residues located in the putative ATP binding site of the P2X1 receptor. J. Neurosci. 27, 1456–1466. (doi:10.1523/JNEUROSCI.3105-06.2007) [PubMed]
78. Nagaya N, Tittle RK, Saar N, Dellal SS, Hume RI 2005. An intersubunit zinc binding site in rat P2X2 receptors. J. Biol. Chem. 280, 25 982–25 993. (doi:10.1074/jbc.M504545200) [PMC free article] [PubMed]
79. Jiang R, Lemoine D, Martz A, Taly A, Gonin S, Prado de Carvalho L, Specht A, Grutter T 2011. Agonist trapped in ATP-binding sites of the P2X2 receptor. Proc. Natl Acad. Sci. USA 108, 9066–9071. (doi:10.1073/pnas.1102170108) [PubMed]
80. Marquez-Klaka B, Rettinger J, Nicke A 2009. Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors. Eur. Biophys. J. 38, 329–338. (doi:10.1007/s00249-008-0325-9) [PubMed]
81. Kawate T, Robertson JL, Li M, Silberberg SD, Swartz KJ 2011. Ion access pathway to the transmembrane pore in P2X receptor channels. J. Gen. Physiol. 137, 579–590. (doi:10.1085/jgp.201010593) [PMC free article] [PubMed]
82. Roberts JA, Allsopp RC, El Ajouz S, Vial C, Schmid R, Young MT, Evans RJ 2012. Agonist binding evokes extensive conformational changes in the extracellular domain of the ATP-gated human P2X1 receptor ion channel. Proc. Natl Acad. Sci. USA 109, 4663–4667. (doi:10.1073/pnas.1201872109) [PubMed]
83. Hausmann R, Günther J, Kless A, Kuhlmann D, Kassack MU, Bahrenberg G, Markwardt F, Schmalzing G 2013. Salt bridge switching from Arg290/Glu167 to Arg290/ATP promotes the closed-to-open transition of the P2X2 receptor. Mol. Pharmacol. 83, 73–84. (doi:10.1124/mol.112.081489) [PubMed]
84. Stelmashenko O, Compan V, Browne LE, North RA 2014. Ectodomain movements of an ATP-gated ion channel (P2X2 receptor) probed by disulfide locking. J. Biol. Chem. 289, 9909–9917. (doi:10.1074/jbc.M113.542811) [PMC free article] [PubMed]
85. Lemoine D, et al. 2013. Optical control of an ion channel gate. Proc. Natl Acad. Sci. USA 110, 20 813–20 818. (doi:10.1073/pnas.1318715110) [PubMed]
86. Browne LE, Nunes JP, Sim JA, Chudasama V, Bragg L, Caddick S, North RA 2014. Optical control of trimeric P2X receptors and acid-sensing ion channels. Proc. Natl Acad. Sci. USA 111, 521–526. (doi:10.1073/pnas.1318582111) [PubMed]
87. Rothwell SW, Stansfeld PJ, Bragg L, Verkhratsky A, North RA 2014. Direct gating of ATP-activated ion channels (P2X2 receptors) by lipophilic attachment at the outer end of the second transmembrane domain. J. Biol. Chem. 289, 618–626. (doi:10.1074/jbc.M113.529099) [PMC free article] [PubMed]
88. Werner P, Seward EP, Buell GN, North RA 1996. Domains of P2X receptors involved in desensitization. Proc. Natl Acad. Sci. USA 93, 15 485–15 490. (doi:10.1073/pnas.93.26.15485) [PubMed]
89. Allsopp RC, Evans RJ 2015. Contribution of the juxtamembrane intracellular regions to the time course and permeation of ATP-gated P2X7 receptor ion channels. J. Biol. Chem. 290, 14 556–14 566. (doi:10.1074/jbc.M115.642033) [PMC free article] [PubMed]
90. Browne LE, Compan V, Bragg L, North RA 2013. P2X7 receptor channels allow direct permeation of nanometer-sized dyes. J. Neurosci. 33, 3557–3566. (doi:10.1523/JNEUROSCI.2235-12.2013) [PubMed]
91. Baconguis I, Gouaux E 2012. Structural plasticity and dynamic selectivity of acid-sensing ion channel–spider toxin complexes. Nature 489, 400–405. (doi:10.1038/nature11375) [PMC free article] [PubMed]
92. Chrovian CC, Rech JC, Bhattacharya A, Letavic MA 2014. P2X7 antagonists as potential therapeutic agents for the treatment of CNS disorders. Prog. Med. Chem. 53, 65–100. (doi:10.1016/B978-0-444-63380-4.00002-0) [PubMed]
93. Bhattacharya A, et al. 2013. Pharmacological characterization of a novel centrally permeable P2X7 receptor antagonist: JNJ-47965567. Br. J. Pharmacol. 170, 624–640. (doi:10.1111/bph.12314) [PMC free article] [PubMed]
94. Ferrari D, Chiozzi P, Falzoni S, Dal Susino M, Melchiorri L, Baricordi OR, Di Virgilio F 1997. Extracellular ATP triggers IL-1 beta release by activating the purinergic P2Z receptor of human macrophages. J. Immunol. 159, 1451–1458. [PubMed]
95. Romagnoli R, Baraldi PG, Cruz-Lopez O, Lopez-Cara C, Preti D, Borea PA, Gessi S 2008. The P2X7 receptor as a therapeutic target. Expert Opin. Ther. Targets 12, 647–661. (doi:10.1517/14728222.12.5.647) [PubMed]
96. North RA, Jarvis MF 2013. P2X receptors as drug targets. Mol. Pharmacol. 83, 759–769. (doi:10.1124/mol.112.083758) [PubMed]
97. Keystone EC, Wang MM, Layton M, Hollis S, McInnes IB 2012. Clinical evaluation of the efficacy of the P2X7 purinergic receptor antagonist AZD9056 on the signs and symptoms of rheumatoid arthritis in patients with active disease despite treatment with methotrexate or sulphasalazine. Annu. Rheum. Dis. 71, 1630–1635. (doi:10.1136/annrheumdis-2011-143578) [PubMed]
98. Stock TC, Bloom BJ, Wei N, Ishaq S, Park W, Wang X, Gupta P, Mebus CA 2012. Efficacy and safety of CE-224,535, an antagonist of P2X7 receptor, in treatment of patients with rheumatoid arthritis inadequately controlled by methotrexate. J. Rheumatol. 39, 720–727. (doi:10.3899/jrheum.110874) [PubMed]
99. Burnstock G. 2009. Purinergic mechanosensory transduction and visceral pain. Mol. Pain 5, 69–71. (doi:10.1186/1744-8069-5-69) [PMC free article] [PubMed]
100. Abdulqawi R, Dockry R, Holt K, Layton G, McCarthy BG, Ford AP, Smith JA 2015. P2X3 receptor antagonist (AF-219) in refractory chronic cough: a randomised, double-blind, placebo-controlled phase 2 study. Lancet 385, 1198–1205. (doi:10.1016/S0140-6736(14)61255-1) [PubMed]
101. Belvisi MG. 2015. Therapeutic advances for treatment-resistant cough. Lancet 385, 1160–1162. (doi:10.1016/S0140-6736(14)61740-2) [PubMed]
102. Mulryan K, et al. 2000. Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors. Nature 403, 86–89. (doi:10.1038/47495) [PubMed]
103. White CW, Choong YT, Short JL, Exintaris B, Malone DT, Allen AM, Evans RJ, Ventura S 2013. Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice. Proc. Natl Acad. Sci. USA 110, 20 825–20 830. (doi:10.1073/pnas.1318624110) [PubMed]

Articles from Philosophical Transactions of the Royal Society B: Biological Sciences are provided here courtesy of The Royal Society