P2X receptors.

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 1ß. 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 Ca(2+) and ATP together to control life and death'.

Ectodomain movements of an ATP-gated ion channel (P2X2 receptor) probed by disulfide locking.

The ectodomain of the P2X receptor is formed mainly from two- or three-stranded ß-sheets provided symmetrically by each of the three subunits. These enclose a central cavity that is closed off furthest from the plasma membrane (the turret) and that joins with the transmembrane helices to form the ion permeation pathway. Comparison of closed and open crystal structures indicates that ATP binds in a pocket positioned between strands provided by different subunits and that this flexes the ß-sheets of the lower body and enlarges the central cavity: this pulls apart the outer ends of the transmembrane helices and thereby opens an aperture, or gate, where they intersect within the membrane bilayer. In the present work, we examined this opening model by introducing pairs of cysteines into the rat P2X2 receptor that might form disulfide bonds within or between subunits. Receptors were expressed in human embryonic kidney cells, and disulfide formation was assessed by observing the effect of dithiothreitol on currents evoked by ATP. Substitutions in the turret (P90C, P89C/S97C), body wall (S65C/S190C, S65C/D315C) and the transmembrane domains (V48C/I328C, V51C/I328C, S54C/I328C) strongly inhibited ATP-evoked currents prior to reduction with dithiothreitol. Western blotting showed that these channels also formed predominately as dimers and/or trimers rather than monomers. The results strongly support the channel opening mechanism proposed on the basis of available crystal structures.

Optical control of trimeric P2X receptors and acid-sensing ion channels.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Direct gating of ATP-activated ion channels (P2X2 receptors) by lipophilic attachment at the outer end of the second transmembrane domain.

The ionic pore of the P2X receptor passes through the central axis of six transmembrane (TM) helices, two from each of three subunits. Val(48) and Ile(328) are at the outer end of TM1 and TM2, respectively. Homology models of the open and closed states of P2X2 indicate that pore opening is associated with a large lateral displacement of Ile(328). In addition, molecular dynamics simulations suggest that lipids enter the interstices between the outer ends of the TM domains. The P2X2(I328C) receptor was activated by propyl-methanethiosulfonate (MTS) as effectively as by ATP, but cysteine substitutions elsewhere in TM2 had no such effect. Other lipophilic MTS compounds (methyl, ethyl, and tert-butylethyl) had a similar effect but not polar MTS. The properties of the conducting pathway opened by covalent attachment of propyl-MTS were the same as those opened by ATP, with respect to unitary conductance, rectification, and permeability of N-methyl-d-glucamine. The ATP-binding residue Lys(69) was not required for the action of propyl-MTS, although propyl-MTS did not open P2X2(K308A/I328C) receptors. The propyl-MTS did not open P2X2 receptors in which the Val(48) side chain was removed (P2X2(V48G/I328C)). The results suggest that an interaction between Val(48) and Ile(328) stabilizes the closed channel and that this is broken by covalent attachment of a larger lipophilic moiety at the I328C receptors. Lipid intercalation between the separating TM domains during channel opening would be facilitated in P2X2(I328C) receptors with attached propyl-MTS. The results are consistent with the channel opening mechanism proposed on the basis of closed and open crystal structures and permit the refinement of the position of the TMs within the bilayer.

Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel.

Rab GTPases play key roles in the delivery, docking and fusion of intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase activity is controlled is poorly understood. Here we describe a mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. We show that activation of P2XA, an intracellular ion channel localized to the Dictyostelium discoideum contractile vacuole system, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Vacuole fusion and Rab11a downregulation require the activity of CnrF, an EF-hand-containing Rab GAP found in a complex with Rab11a and P2XA. CnrF Rab GAP activity for Rab11a is enhanced by the presence of calcium and the EF-hand domain. These findings suggest that P2XA activation results in vacuolar calcium release, which triggers activation of CnrF Rab GAP activity and subsequent downregulation of Rab11a to allow vacuole fusion.

P2X receptor intermediate activation states have altered nucleotide selectivity.

Purinergic P2X receptors are widely distributed in the nervous system and are known to play roles in primary afferent transmission and central respiratory regulation. They are trimeric membrane proteins, with the extracellular domain that provides three intersubunit ATP binding sites. We expressed the rat P2X7 receptor in human embryonic kidney cells and measured membrane currents before and after photo-affinity labeling with the agonist 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP). After tethering BzATP with ultraviolet light, a persistent current remained after washing out the agonist. Additional current could now be elicited by other nucleotides (CTP and ADP) that are not normally effective as P2X receptor agonists. Similar results were obtained at P2X2 receptors even without previous agonist tethering: exposure to low concentrations of ATP caused the receptor to become sensitive to activation by CTP and ADP. The results show that ATP binding to the first of the three binding sites causes a conformational change to an intermediate closed state that shows increased effectiveness of pyrimidine and diphosphate nucleotide analogs.

Functional properties of five Dictyostelium discoideum P2X receptors.

The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. 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 impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.

P2X7 receptor channels allow direct permeation of nanometer-sized dyes.

P2X receptors are widely distributed in the nervous system, and P2X7 receptors have roles in neuropathic pain and in the release of cytokines from microglia. They are trimeric membrane proteins, which open an integral ion channel when ligated by extracellular ATP. This channel is preferentially permeable to small cations (sodium, potassium, calcium) but also allows permeation of larger cations such as N-methyl-d-glucamine. ATP also leads to entry of fluorescent dyes in many cells expressing P2X7 receptors, but controversy persists as to whether such large molecules pass directly through the open ion channel or enter the cell by a different route. We measured cellular fluorescence and membrane currents in individual human embryonic kidney cells expressing rat P2X7 receptors. Introduction of positive side chains by mutagenesis into the inner half of the pore-forming second transmembrane domain of the receptor (T348K, D352N, D352K) increased relative permeability of chloride ions. It also promoted entry of the large (>1 nm) negative dye fluorescein-5-isothiocyanate while decreasing entry of the structurally similar but positive dye ethidium. Furthermore, larger cysteine-reactive methanethiosulfonates [sulforhodamine-methanethiosulfonate and 2-((biotinoyl)amino)ethyl methanethiosulfonate] reduced both ATP-evoked currents and dye entry when applied to open P2X7[G345C] receptors. The results demonstrate that the open channel of the P2X7 receptor can allow passage of molecules with sizes up to 1.4 nm.

P2X receptors as drug targets.

The study of P2X receptors has long been handicapped by a poverty of small-molecule tools that serve as selective agonists and antagonists. There has been progress, particularly in the past 10 years, as cell-based high-throughput screening methods were applied, together with large chemical libraries. This has delivered some drug-like molecules in several chemical classes that selectively target P2X1, P2X3, or P2X7 receptors. Some of these are, or have been, in clinical trials for rheumatoid arthritis, pain, and cough. Current preclinical research programs are studying P2X receptor involvement in pain, inflammation, osteoporosis, multiple sclerosis, spinal cord injury, and bladder dysfunction. The determination of the atomic structure of P2X receptors in closed and open (ATP-bound) states by X-ray crystallography is now allowing new approaches by molecular modeling. This is supported by a large body of previous work using mutagenesis and functional expression, and is now being supplemented by molecular dynamic simulations and in silico ligand docking. These approaches should lead to P2X receptors soon taking their place alongside other ion channel proteins as therapeutically important drug targets.

Neuromodulation by extracellular ATP and P2X receptors in the CNS.

Extracellular adenosine 5' triphosphate (ATP) is a widespread cell-to-cell signaling molecule in the brain, where it activates cell surface P2X and P2Y receptors. P2X receptors define a protein family unlike other neurotransmitter-gated ion channels in terms of sequence, subunit topology, assembly, and architecture. Within milliseconds of binding ATP, they catalyze the opening of a cation-selective pore. However, recent data show that P2X receptors often underlie neuromodulatory responses on slower time scales of seconds or longer. Herein, we review these findings at molecular, cellular and systems levels. We propose that, while P2X receptors are fast ligand-gated cation channels, they are most adept at mediating slow neuromodulatory functions that are more widespread and more physiologically utilized than fast ATP synaptic transmission in the CNS.

Activation of trimeric P2X2 receptors by fewer than three ATP molecules.

P2X receptors are trimeric membrane proteins. When they bind extracellular ATP, a conformational change occurs that opens a transmembrane ion channel. The ATP-binding pocket is formed in a cleft between two subunits, and a critical amino acid residue for ATP contact is Lys69 (P2X2 numbering). In the present work, we sought to determine whether the binding of fewer than three ATP molecules could open the ion channel. We expressed eight concatenated cDNAs in human embryonic kidney cells, which encoded three serially joined, epitope-tagged, subunits with either Lys or Ala at position 69 (denoted as KKK, KKA, KAK, AKK, KAA, AKA, AAK, and AAA). Western blotting of surface-biotinylated proteins indicated that breakdown of concatemers to individual subunits was minimal. Recording of membrane currents in response to ATP (whole cell and excised outside-out patch) showed that all formed functional channels except AAK, AKA, and AAA. There was no difference in the kinetics of activation and deactivation among KKK, KKA, KAK, and AKK channels, and amplitude of the unitary conductances was in all cases not different from that found after expression of a single wild-type subunit. Currents through KKA and KAK receptors were larger than those observed for AKK receptors. The results indicate that trimeric P2X receptors containing only two intact binding sites can be readily activated by ATP.

Pannexin 1 forms an anion-selective channel.

Pannexin 1 (Panx1) is expressed in various mammalian tissues including the brain and immune cells. Here, we present evidence that Panx1 when expressed in mammalian cells, forms anion-selective channels, with a rank order of permeabilities: NO (3) (-)> I(-) > Br (-)> Cl (-) > F (-)>> aspartate (-)≈ glutamate (-)≈ gluconate(-). Single-channel Panx1-mediated currents have a unitary conductance around 68 pS. Our results show that Panx1 assembles into a membrane anion channel with a relatively low single-channel conductance.

Role of P2X4 receptors in synaptic strengthening in mouse CA1 hippocampal neurons.

P2X4 receptors are calcium-permeable cation channels gated by extracellular ATP. They are found close to subsynaptic sites on hippocampal CA1 neurons. We compared features of synaptic strengthening between wild-type and P2X4 knockout mice (21-26 days old). Potentiation evoked by a tetanic presynaptic stimulus (100 Hz, 1 s) paired with postsynaptic depolarization was less in P2X4(-/-) mice than in wild-type mice (230 vs. 50% potentiation). Paired-pulse ratios and the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) were not different between wild-type and knockout mice. Prior hyperpolarization (ten 3 s pulses to -120 mV at 0.17 Hz) potentiated the amplitude of spontaneous EPSCs in wild-type mice, but not in P2X4(-/-) mice; this potentiation was not affected by nifedipine, but was abolished by 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) in the recording pipette. The amplitude of N-methyl-d-aspartate EPSCs (in 6-cyano-7-nitroquinoxaline-2,3-dione, 10 or 30 µm, at -100 mV) facilitated during 20 min recording in magnesium-free solution. In wild-type mice, this facilitation of the N-methyl-d-aspartate EPSC was reduced by about 50% by intracellular BAPTA (10 mM), ifenprodil (3 µm) or 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 µm). In P2X4(-/-) mice, the facilitation was much less, and was unaffected by intracellular BAPTA, ifenprodil (3 µm) or mitogen-activated protein (MAP) kinase inhibitor 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 µm). This suggests that the absence of P2X4 receptors limits the incorporation of NR2B subunits into synaptic N-methyl-d-aspartate receptors.

P2X receptor channels show threefold symmetry in ionic charge selectivity and unitary conductance.

In the closed structure of the P2X cation channel, three α-helical transmembrane domains cross the membrane obliquely. In rat P2X2 receptors, these intersect at Thr(339). Replacing Thr(339) by lysine in one, two or three subunits progressively increased chloride permeability and reduced unitary conductance. This implies that the closed-open transition involves a symmetrical separation of the three subunits and that Thr(339) from each subunit contributes symmetrically to the open channel permeation pathway.

New structure enlivens interest in P2X receptors.

P2X receptors are ATP-gated membrane ion channels with multifarious roles, including afferent sensation, autocrine feedback loops, and inflammation. Their molecular operation has been less well elucidated compared with other ligand-gated channels (nicotinic acetylcholine receptors, ionotropic glutamate receptors). This will change with the recent publication of the crystal structure of a closed P2X receptor. Here we re-interpret results from 15 years of experiments using site-directed mutagenesis with a model based on the new structure. Previous predictions of receptor stoichiometry, the extracellular ATP binding site, inter-subunit contacts, and many details of the permeation pathway fall into place in three dimensions. We can therefore quickly understand how the channel operates at the molecular level. This is important not only for ion- channel aficionados, but also those engaged in developing effective antagonists at P2X receptors for potential therapeutic use.

Polar residues in the second transmembrane domain of the rat P2X2 receptor that affect spontaneous gating, unitary conductance, and rectification.

Membrane ion channels activated by extracellular ATP (P2X receptors) are widely distributed in the nervous system. Their molecular architecture is fundamentally distinct from that of the nicotinic or glutamate receptor families. We have measured single-channel currents, spontaneous gating, and rectification of rat P2X2 receptor in which polar and charged residues of the second transmembrane domain (TM2) were systematically probed by mutagenesis. The results suggest that Asn(333) and Asp(349) lie respectively in external and internal vestibules. Substitutions at Asn(333), Thr(336), and Ser(340) were particularly likely to cause spontaneously active channels. At Thr(336), Thr(339), and Ser(340), the introduction of positive charge (Arg, Lys, or His, or Cys followed by treatment with 2-aminoethyl methanethiosulphonate) greatly enhanced outward currents, suggesting that side-chains of these three residues are exposed in the permeation pathway of the open channel. These functional findings are interpreted in the context of the recently reported 3.1 A crystal structure of the zebrafish P2X4.1 receptor in the closed state. They imply that the gate is formed by residues Asn(333) to Thr(339) and that channel opening involves a counter-clockwise rotation and separation of the TM2 helices.

Electrophysiological and morphological properties of neurons in the substantia gelatinosa of the mouse trigeminal subnucleus caudalis.

The excitability of the second order neurons within the trigeminal subnucleus caudalis underlies pain perception and processing in migraine and trigeminal neuralgia. These neurons were studied with whole-cell patch-clamp technique in slices from mouse brain stem. Electrical and morphological characteristics of 56 neurons were determined. Four categories were distinguished from electrophysiological properties: tonic (39%), phasic (34%), delayed (16%) and single spiking (11%). These categories did not show distinct morphological properties. Neurons had tetrodotoxin-sensitive sodium currents that activated and inactivated within milliseconds. They also showed a high voltage-activated, slowly inactivating calcium current: up to half of this current was blocked by omega-conotoxin GVIA (1microM) and omega-agatoxin IVA (100-300 nM), but it was not affected by nifedipine (10microM). Exogenously applied capsaicin (1microM) and alphabetamethylene-5'-adenosine triphosphate (100microM) elicited large amplitude, spontaneous excitatory postsynaptic currents that were blocked by capsazepine (10microM) and 5-[(3-phenoxybenzyl)-(1,2,3,4-tetrahydro-naphthalen-1-yl)-carbamoyl]-benzene-1,2,4-tricarboxylic acid (A-317491: 10microM), respectively. Thus, neurons of the mouse trigeminal subnucleus caudalis substantia gelatinosa exhibit N-type and P/Q-type voltage-gated calcium channels, and receive presynaptic afferents that express TRPV1 and P2X(2/3) receptors. These results suggest possible therapeutic interventions, and serve as a basis for the characterization of cellular changes that may underlie trigeminal neuropathic pain.

Signaling at purinergic P2X receptors.

P2X receptors are membrane cation channels gated by extracellular ATP. Seven P2X receptor subunits (P2X(1-7)) are widely distributed in excitable and nonexcitable cells of vertebrates. They play key roles in inter alia afferent signaling (including pain), regulation of renal blood flow, vascular endothelium, and inflammatory responses. We summarize the evidence for these and other roles, emphasizing experimental work with selective receptor antagonists or with knockout mice. The receptors are trimeric membrane proteins: Studies of the biophysical properties of mutated subunits expressed in heterologous cells have indicated parts of the subunits involved in ATP binding, ion permeation (including calcium permeability), and membrane trafficking. We review our current understanding of the molecular properties of P2X receptors, including how this understanding is informed by the identification of distantly related P2X receptors in simple eukaryotes.

Ectodomain lysines and suramin block of P2X1 receptors.

P2X(1) receptors belong to a family of cation channels gated by extracellular ATP; they are found inter alia in smooth muscle, platelets, and immune cells. Suramin has been widely used as an antagonist at P2X receptors, and its analog 4,4',4'',4'''-[carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino))] tetrakis-benzene-1,3-disulfonic acid (NF449) is selective for the P2X(1) subtype. Human and mouse P2X(1) receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP were recorded. ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors. Suramin (10 microm) and NF449 (3-300 nM) effectively blocked the human receptor. Suramin had little effect on the mouse receptor. Suramin and NF449 are polysulfonates, with six and eight negative charges, respectively. We hypothesized that species differences might result from differences in positive residues presented by the large receptor ectodomain. Four lysines in the human sequence (Lys(111), Lys(127), Lys(138), and Lys(148)) were changed individually and together to their counterparts in the mouse sequence. The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449. Conversely, when lysine was introduced into the mouse receptor, the sensitivity to block by suramin and NF449 was much increased for E138K, but not for Q111K, Q127K, or N148K. The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

Molecular shape, architecture, and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.

P2X receptors are ATP-gated nonselective cation channels with important physiological roles. However, their structures are poorly understood. Here, we analyzed the architecture of P2X receptors using fluorescence resonance energy transfer (FRET) microscopy and direct structure determination using electron microscopy. FRET efficiency measurements indicated that the distance between the C-terminal tails of P2X(4) receptors was 5.6 nm. Single particle analysis of purified P2X(4) receptors was used to determine the three-dimensional structure at a resolution of 21A; the orientation of the particle with respect to the membrane was assigned by labeling the intracellular C termini with 1.8-nm gold particles and the carbohydrate-rich ectodomain with lectin. We found that human P2X(4) is a globular torpedo-like molecule with an approximate volume of 270 nm(3) and a compact propeller-shaped ectodomain. In this structure, the distance between the centers of the gold particles was 6.1 nm, which closely matches FRET data. Thus, our data provide the first views of the architecture, shape, and size of single P2X receptors, furthering our understanding of this important family of ligand-gated ion channels.