ATP-gated cation channel (purinoceptor or ATP-neuroreceptor). Residues Glu52-Gly96 play roles in agonist binding and channel gating (Allsopp et al., 2011).  The rat protein is 89% identical to the human ortholog. Mutations likely to confer ivermectin sensitivity to human P2X1 have been proposed (Pasqualetto et al. 2018).  The P2X1 receptor is a trimeric ligand-gated ion channel that plays an important role in urogenital and immune functions. Bennetts et al. 2024 employed cryogenic electron microscopy (cryo-EM) to elucidate the structures of the P2X1 receptor in an ATP-bound desensitised state and an NF449-bound closed state. NF449, a potent P2X1 receptor antagonist, engages the receptor distinctively, while ATP, the endogenous ligand, binds in a manner consistent with other P2X receptors. To explore the molecular basis of receptor inhibition, activation, and ligand interactions, key residues involved in ligand and metal ion binding were mutated. Radioligand binding assays with [³H]-α,β-methylene ATP and intracellular calcium ion influx assays were used to evaluate the effects of these mutations. These experiments validated key ligand-receptor interactions and identified conserved and non-conserved residues critical for ligand binding or receptor modulation (Bennetts et al. 2024). 

Animals

P2X1 of Homo sapiens

Green algal ATP-gated cation channel receptor P2X4 of 384 aas, 2 TMSs (Fountain et al., 2008).

Eukaryotes

P2X4 of Ostreococcus lucimarinus

P2X5 ATP-activated receptor, P2X5R or P2RX5, of 422 aas and 2 TMSs, N- and C-terminal (Sun et al. 2019). This receptor can transport both cations and anions, in contrast to most other P2X channels (Tam et al. 2023).

P2X5R of Homo sapiens

P2X7 purinoceptor of 595 aas and 2 TMSs. All residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptors, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. Rupert et al. 2020 examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. The K49R, F322Y, F322W, and F322L mutants reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. Thus, clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel (Rupert et al. 2020). This protein is 80% identical to the human ortholog (TC# 1.A.7.1.3). The P2X7 receptor in normal and cancer cells, in the perspective of nucleotide signaling, has been reviewed (Matyśniak et al. 2022). N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes. The improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes (Aquino et al. 2022). The large intracellular C-terminus of the pro-inflammatory P2X7 ion channel receptor (P2X7R) is associated with diverse P2X7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2X7R identified a membrane-associated anchoring domain, an open-state stabilizing 'cap' domain, and a globular 'ballast domain' containing GTP/GDP and dinuclear Zn²⁺-binding sites with unknown functions. To investigate protein dynamics during channel activation, Durner et al. 2023 incorporated the environment-sensitive fluorescent unnatural amino acid, L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) into Xenopus laevis oocyte-expressed P2X7Rs and performed voltage clamp fluorometry (VCF). Predicted conformational changes within the extracellular and the transmembrane domains were confirmed. The ballast domain functions fairly independently of the extracellular ATP binding domain and may require activation by additional ligands and/or protein interactions (Durner et al. 2023).  The P2X7 receptor provides a mechanistic biomarker for epilepsy (Engel 2023). Puerarin inhibits NLRP3-caspase-1-GSDMD-mediated pyroptosis via the P2X7 receptor in cardiomyocytes and macrophages (Sun et al. 2023).

P2X7 of Rattus norvegicus (Rat)

ATP-gated cation channel (purinoceptor or ATP-neuroreceptor), P2X2.  His33 and Ser345 are proximal to each other across an intra-subunit interface, and the relative movement between the two TMSs is likely important for transmitting the action of ATP binding to the opening of the channel (Liang et al. 2013).  Two processes contribute to receptor desensitization, one, bath calcium-independent and the other, bath calcium-dependent, the latter being more important (Coddou et al. 2015).  ATP dissociation causes reduction in outer pore expansion compared to the ATP-bound state. Moreover, the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion (Habermacher et al. 2016). Hearing loss mutations alter the functional properties of human P2X2 receptor channels through more than one mechanism (George et al. 2019). Residues in TMSs of the P2X4 receptor that contribute to channel function and ethanol sensitivity have been identified (Popova et al. 2020). Self-assembly of mammalian cell membranes on bioelectronic devices with  P2X2 channel has been achieved (Liu et al. 2020). Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating (Sivcev et al. 2020).

Animals

P2X2 of Rattus norvegicus

ATP-gated NaCl-regulated nonselective cation (Na⁺, K⁺ and Ca²⁺) channel, the P2X purinoreceptor 7, P2X7, P2RX7 or P2X7R. It expands to accommodate large molecules such as NAD, N-methyl-D-glucamine and triethyl ammonium) (Li et al., 2005; Lu et al., 2007) and plays a role in changing pain thresholds. A region called ADSEG in all P2X receptors is located in the M2 domain which aligns with TMS 5 in VIC K⁺ channels (1.A.1). ADSEG from P2X(7)R forms cation-selective channels in artificial lipid bilayers and biological membranes similar to those of the full length protein (de Souza et al., 2011). Channel activity is regulated by calmodulin (Roger et al., 2008).  P2XRs allow direct permeation of nanometer-sized dyes (Browne et al. 2013).  Macrophage P2X7 receptors are modulated in response to infection with Leishmania amazonensis so that they become more permeable to anions and less permeable to cations (Marques-da-Silva et al. 2011).  Residues involved in pore conductivity and agonist sensitivity have been identified (Jindrichova et al. 2015) as have residues involved in channel activation (Caseley et al. 2016). The channel opening extends from the pre-TMS2 region through the outer half of the trihelical TMS2 channel; the gate and the selectivity filter have been identified (Pippel et al. 2017). The purinergic receptors, P2RX4 and P2RX7, when mutated, affect susceptibility to multiple sclerosis (MS) (Sadovnick et al. 2017). P2X7 may serve as a receptor for the regulation of annexin secretion during macrophage polarization (de Torre-Minguela et al. 2016). These receptors can reduce salivary gland inflammation (Khalafalla et al. 2017). The P2X7 receptor forms ion channels dependent on lipids but independently of its cytoplasmic domain (Karasawa et al. 2017).  A truncated naturally occurring variant of P2X7, P2X7-j of 258 aas, lacks the entire intracellular carboxyl terminus, the second TMS, and the distal third of the extracellular loops of the full-length P2X7 receptor. P2X7-j, expressed in the plasma membrane, failed to form pores and mediate apoptosis (Feng et al. 2006). P2X7-j formed heterooligomers with and blocked P2X7-mediated channel formation. Alternative splicing of P2X7 controls gating of the ion channel by ADP-ribosylation (Schwarz et al. 2012). Three distinct roles for P2X7 during adult neurogenesis have been demonstrated, and these depend on the extracellular ATP concentrations: (i) P2X7 receptors can form transmembrane pores leading to cell death, (ii) P2X7 receptors can regulate rates of proliferation, likely via calcium signalling, and (iii) P2X7 can function as scavenger receptors in the absence of ATP, allowing neural progenitor cells (NPCs) to phagocytose apoptotic NPCs during neurogenesis (Leeson et al. 2018). P2X7 also plays a role in purinergic vasotoxicity and cell death (Shibata et al. 2018). NAD⁺ covalently modifies the P2X7R of mouse T lymphocytes, thus lowering the ATP threshold for activation. Other structurally unrelated agents have been reported to activate  P2X7R: (a) the antibiotic polymyxin B, possibly a positive allosteric P2X7R modulator, (b) the bactericidal peptide LL-37, (c) the amyloidogenic β peptide, and (d) serum amyloid A (Di Virgilio et al. 2018). Some agents, such as Alu-RNA, have been suggested to activate P2X7R, acting on the intracellular N- or C-terminal domains. P2X7R of enteric neurons may be involved in diabetes-induced nitrous oxide (NOS) neuron damage via combining with pannexin-1 to form transmembrane pores which transport macromolecular substances and calcium into the cells (Zhang et al. 2019). ATP-gated P2X7 receptors require chloride channels to promote inflammation in human macrophages (Janks et al. 2019). P2X7 overexpression is can be associated with cancer progression. P2X7 plays also an important role in glioma biology (Matyśniak et al. 2020). Upon activation by its main ligand, extracellular ATP, P2X7 can form a nonselective channel for cations to enter the cell, but prolonged activation, via high levels of extracellular ATP over an extended time period can lead to the formation of a macropore, leading to depolarization of the plasma membrane and ultimately to cell death. Thus, dependent on its activation state, P2X7 can either drive cell survival and proliferation, or induce cell death. It is relevant to cancerous growth (Lara et al. 2020). The human P2X7 receptor is a ligand gated ion channel opened by binding of ATP, like the other P2X receptor subtypes. P2X7 receptors become activated under pathological conditions of ATP release like hypoxia or cell destruction. They are involved in inflammatory and nociceptive reactions of the organism to these pathological events. Polar residues of the second TMS of the three protein subunits are important for ion conduction, with S342 constituting the ion selectivity filter and the gate of the channel. The specific long C-terminal domains are important for hP2X7 receptor ion channel function, as their loss strongly decreases ion channel currents (Markwardt 2020). Studies of the enhancement of P2X(7)-induced pore formation and apoptosis revealed an early effect of diabetes on retinal microvasculature; diabetes appears to facilitate the channel-to-pore transition that occurs during activation of these purinoceptors (Sugiyama et al. 2004). Regorafenib exhibits antitumor activity on the breast cancer cell line via modulation of the P2X7/HIF-1alpha/VEGF, P2X7/P38, P2X7/ERK/NF-kappaB, and P2X7/beclin 1 pathways (Salahuddin et al. 2021). The involvement of the P2RX7 purinoreceptor in triggering mitochondrial dysfunction during the development of neurodegenerative disorders has been reviewed (Zelentsova et al. 2022). The P2X7 receptor and purinergic signaling play roles in orchestrating mitochondrial dysfunction in neurodegenerative diseases (Zelentsova et al. 2022). The P2X7 purinergic receptor represents a potential target in heart diseases (Bin Dayel et al. 2023). Conserved and receptor specific TMS1 residues control surface expression of the P2X7 protein, nonpolar residues control receptor sensitization, and D48 regulates intrinsic channel properties (Rupert et al. 2023). The P2X7 receptor of microglia in the olfactory bulb mediates the pathogenesis of olfactory dysfunction in a mouse model of allergic rhinitis (Ren et al. 2023). P2RX7 variants interact with distal and more etiological stressors in influencing the severity of anxiety symptoms (Kristof et al. 2023). P2X7 receptor inhibition ameliorates ubiquitin-proteasome system dysfunction associated with Alzheimer's disease (Bianchi et al. 2023). P2X7R radioligands are reliable tools for the diagnosis of neuroinflammation in clinical studies, and detection and measurement of free P2X7 receptor (or the P2X7 subunit) in human blood suggested its potential use as a circulating marker of inflammation (Di Virgilio et al. 2023). There are  three frequent coding polymorphisms in the gene for the human P2X7 ion channel, and their functions are known (Schäfer et al. 2022). A P2X7 receptor blockade reduces pyroptotic inflammation and promotes phagocytosis in Vibrio vulnificus infection (Wann et al. 2023). Niemann-Pick disease type C is a rare autosomal recessive of lysosomal storage disorder characterized by impaired intracellular lipid transport and has a tendency to accumulate the fatty acids and glycosphingolipids in a variety of neurovisceral tissues, and the mutational impact in causing Niemann-Pick disease type C has been studied (Kannan et al. 2023). Receptor agonists and antagonists and other modulators of purinergic signalling have potential as novel therapeutics for a broad range of diseases and conditions. An up-to-date description of selected efforts to discover and develop new small molecular purinergic drugs has appeared (Jacobson and Salvemini 2023). Astrocytes induce ischemic tolerance via P2X7 receptor-mediated lactate release (Hirayama et al. 2024). P2X7 receptors in dendritic cells and macrophages have implications in antigen presentation and T lymphocyte activation (Acuña-Castillo et al. 2024).  Huang et al. demonstrate that the complex of sodium/potassium-transporting ATPase subunit alpha (NKAα1) and purinergic P2X7 receptor (P2X7R) maintains the resting state of microglial membranes. Stress increases free P2X7R that then binds to ATP to activate microglia, which may promote anxious behaviors (Fang and Lai 2024).

Animals

P2X7 of Homo sapiens (Q99572)

The P2X4 receptor (P2X4R) of the zebrafish of 389 aas and 2 TMSs. The 3-d structure is known in its closed, resting state (Kawate et al., 2009).  A hift of L340 packing between different sites may alter the side-chain orientation that frees or occludes the pore. L340, A344 and A347 may also gate the pore by a expansion-contraction mechanism (Li 2015). Ivermectin binds to the transmembrane domain while Zn²⁺ binds to the extracellular domain, but they exhbit additive cooperativity (Latapiat et al. 2017).

Animals

P2X(4) purinoceptor (ATP) gated ionotropic receptor, subunit 4 of Danio rerio (Q98TZ0)

The purinergic receptor, P2X4, is sensitive to the macrocyclic lactone, ivermectin, which allosterically modulates both ion conduction and channel gating (Samways et al., 2012). The secondary structure and gating rearrangements of TMSs in rat P2X4 receptor channels have been proposed (Silberberg et al. 2005). Bile acids inhibit the human P2X4 (Ilyaskin et al. 2019). The gating mechanism has been discussed (Du et al., 2012) and considered to be determined by the conformation of the transmembrane domain (Minato et al. 2016; Pierdominici-Sottile et al. 2016). The crystal structure of the ATP-gated P2X4 ion channel in the closed state has been reported (Kawate et al., 2009). Unobstructed lateral portals are preferentially used as access routes to the pores of P2X receptors (Samways et al., 2011).  Activation is ATP-dependent and rapid, but desensitization occurs within seconds and is ATP-independent (Stojilkovic et al. 2010). Ectodomain cysteines play roles in agonist binding and channel gating (Rokic et al. 2010).  Evermectin has distinct effects on opening and dilation of the channel pore, the first accounting for increased peak current amplitude, and the latter correlating with changes in the kinetics of receptor deactivation (Zemkova et al. 2014). Conserved amino acids within the regions linking the ectodomain with the pore-forming transmembrane domain may contribute to signal transduction and channel gating (Gao et al. 2015; Jelínkova et al. 2008). Binding of ATP produces distortions in the chains that eliminate restrictions on the interchain displacements, leading to the opening of the pore (Pierdominici-Sottile et al. 2016). The purinergic receptors, P2RX4 and P2RX7, affect susceptibility to multiple sclerosis (MS) (Sadovnick et al. 2017). P2X4 modulators are used for the treatment of alcohol use disorders (Reyes-Espinosa et al. 2020). Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating (Sivcev et al. 2020). Therapeutic targeting of the P2X4 receptor and mitochondrial metabolism in clear cell renal carcinoma modelshas been achieved (Rupert et al. 2023). A role for K_ATP channels in cytotoxicity of cells that are primed for a rapid immune response has been reported (Feske et al. 2024).

Animals

P2X4 of Homo sapiens (Q99571)

ATP-gated P2X3 receptor. Tyr-37 stabilizes desensitized states and restricts calcium permeability (Jindrichova et al., 2011).  Exhibits "high affinity desensitization" but slow reactivation from the desensitized state (Giniatullin and Nistri 2013). An endogenous regulator of P2X3 in bladder is the Pirt protein (TC#8.A.64.1.1) Gao et al. 2015).  X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states have been determined (Mansoor et al. 2016). The open state structure harbours an intracellular motif termed the 'cytoplasmic cap', which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. P2X3 receptor antagonism attenuates the progression of heart failure (Lataro et al. 2023).  Standardized Centella asiatica extract ECa 233 alleviates pain hypersensitivity by modulating P2X3 (Wanasuntronwong et al. 2024). When P2X3 is in its apo state, its ICD architecture is fairly ordered rather than an unstructured outward folding, enabling allosteric modulation of the signaling of P2X3 receptors (Lin et al. 2024).

Animals

P2X3 receptor of Homo sapiens (P56373)

P2X purinoceptor

Fish

P2X purinoceptor of Tetaodon nigroviridis

The p2X purinoreceptor 4a of 389 aas and 2 TMSs, P2X4a of 388 aas and 2 TMSs. A splice variant of 361 aas also exists and may form heterotrimers with P2RX4a (Townsend-Nicholson et al. 1999). Plays a role in alcoholism (Franklin et al. 2014). P2RX4 deficiency alleviates allergen-induced airway inflamation (Zech et al. 2016).

P2X4a of Mus musculus (Mouse)

Purinorepector, P2X7 (P2RX7) of 595 aas and 2 TMSs.  The crystal structure in complex with a series of allosteric antagonists were published, giving insight into the mechanism of channel antagonism (Pasqualetto et al. 2018). A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function (Skarratt et al. 2020).

P2X7 of Ailuropoda melanoleuca (Giant panda)

The osmoregulatory intracellular P2X receptor, P2XA gated by ATP (present in the osmoreulatory organelle, the contractile vacuole) (Fountain et al., 2007). One of five P2X receptors in D. discoideum is localized to the contractile vacuole with the ligand binding domain facing the lumen. Plays a role in Ca²⁺ signaling, but also is Cl^- permeable. May function in osmoregulation (Ludlow et al., 2009).  Four of the five receptors operate as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). For the P2XA receptor, ATP was the only effective agonist, but extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed an impaired regulatory volume decrease in hypotonic solution. This phenotype was rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptor which appeared to be inactive (Baines et al. 2013).

Slime mold

P2XA of Dictyostelium discoideum (Q55A88)

Partial seqence of a putative P2XR of 273 aas and 1 TMS at residue 150 in the protein.

Putative P2XR of an archaeon (phyllosphere metagenome)

Uncharacterized protein of 507 aas and 2 TMSs, N- and C-terminal.

UP of Cafeteria roenbergensis

Uncharacterized protein of 521 aas and 5 TMSs in a 3 (N-terminal) + 1 (residues 145 - 165) + 1 (residues 430 - 455).

UP of Capsaspora owczarzaki

Uncharacterized protein of 477 aas and 2 TMSs, near the N- and C-termini.

UP of Polarella glacialis

p2x receptor of 448 aas and 2 TMSs, near the N- and C-termini.

p2x receptor of Chrysochromulina tobinii

Uncharacterized protein of 653 aas and 1 centrally located TMS.  This protein may be a fragment, but shows appreciable sequence identity with members of the P2X family.

UP of an archaeon (phyllosphere metagenome)

Uncharacterized P2X recpetor of 399 aas and 2 TMSs/

UP of Guillardia theta

Uncharacterized P2X receptor of 524 aas and 2 TMSs.

UP of Emiliania huxleyi (Pontosphaera huxleyi)

Uncharacterized protein of 488 aas and 2 TMSs.

UP of Vitrella brassicaformis

P2X receptor E isoform X1 of 397 aas and 2 TMSs, N- and C-terminal.

P2XR of Nematostella vectensis

P2X receptor of 373 aas and 2 TMSs, N- and C-terminal.

P2XR of Planoprotostelium fungivorum

Uncharacterized P2X receptor of 458 aas and 2 TMSs, N- and C-terminal.

UP of Catenaria anguillulae

Uncharacterized protein of 396 aas and 2 TMSs, N- and C-terminal.

UP of Rhizophagus diaphanus

P2X receptor E of 1271 aas and 4 TMSs roughly equidistant from each other, at positions 311, 627, 918, and at the C-terminus. The region showing sequence similarity with other members of the P2XR family is residues 300 to 640, including TMSs 1 and 2 of the 3 distinct TMSs.

P2XR E of Symbiodinium microadriaticum