P2X Receptor Family Research Articles

P2X receptors and modulation of pain transmission: Focus on effects of drugs and compounds used in traditional Chinese medicine

Adenosine triphosphate (ATP) is thought to play an important role in nociceptive transmission or pain signals. ATP is implicated in peripheral pain signaling by acting on P2X receptors. ATP can act on cell bodies of primary afferent fibers. Opening of P2X receptor channels and subsequent membrane depolarization are generally regarded as a key element for extracellular ATP to produce pain. Nociceptive neurons express homomeric P2X 3 as well as heteromeric P2X 2/3 receptors. Both types of channels can be expressed separately or together in individual neuron. Selective antagonists for P2X 3 and/or P2X 2/3 receptors may represent a novel series of useful analgesic drugs. In addition to P2X 3 and P2X 2/3 receptors, other subtypes of the P2X receptor family are also involved in the modulation of nocicpetive transmission (e.g. P2X 4, P2X 7 receptors). Chinese traditional medicine, i.e., tetramethylpyrazine (TMP), sodium ferulate (SF) and puerarin can antagonize the nociceptive or pain transmission mediated by P2X 3 and/or P2X 2/3 receptors in primary afferent neurons. P2X 3 and/or P2X 2/3 receptors are the pharmacological targets of TMP, SF and puerarin for the therapeutic treatment of pain. The myocardial ischemic injury enhanced the sensitization of sympathetic afferent neurons with increased intensity of P2X 3, P2X 2/3 immunoreactivity, protein and mRNA expression in SCG, SG or NG neurons. P2X 3, P2X 2/3 receptors antagonist A-317491 inhibited the transmission of cardiac nociceptive response. Myocardial ischemic nociceptive signaling via P2X 3 and P2X 2/3 receptors in the transmission of rat SCG, SG, NG neuronal circuits induces the sympathoexcitatory reflex to exaggerate myocardial tissue injury. Blocking the nociceptive transmission of SCG, SG, NG neuronal circuits mediated by P2X 3 and P2X 2/3 receptors may improve the cardiac dysfunction. P2X 3 and P2X 2/3 receptors in SCG, SG, NG neurons could be considered as new targets for treating myocardial ischemic injury and cardiac arrhythmia.

Predictions Suggesting a Participation of β-Sheet Configuration in the M2 Domain of the P2X 7 Receptor: A Novel Conformation?

Scanning experiments have shown that the putative TM2 domain of the P2X 7 receptor (P2X 7R) lines the ionic pore. However, none has identified an α-helix structure, the paradigmatic secondary structure of ion channels in mammalian cells. In addition, some researchers have suggested a β-sheet conformation in the TM2 domain of P2X 2. These data led us to investigate a new architecture within the P2X receptor family. P2X 7R is considered an intriguing receptor because its activation induces nonselective large pore formation, in contrast to the majority of other ionic channel proteins in mammals. This receptor has two states: a low-conductance channel (∼10 pS) and a large pore (>400 pS). To our knowledge, one fundamental question remains unanswered: Are the P2X 7R channel and the pore itself the same entity or are they different structures? There are no structural data to help solve this question. Thus, we investigated the hydrophobic M2 domain with the aim of predicting the fitted position and the secondary structure of the TM2 segment from human P2X 7R (hP2X 7R). We provide evidence for a β-sheet conformation, using bioinformatics algorithms and molecular-dynamics simulation in conjunction with circular dichroism in different environments and Fourier transform infrared spectroscopy. In summary, our study suggests the possibility that a segment composed of residues from part of the M2 domain and part of the putative TM2 segment of P2X 7R is partially folded in a β-sheet conformation, and may play an important role in channel/pore formation associated with P2X 7R activation. It is important to note that most nonselective large pores have a transmembrane β-sheet conformation. Thus, this study may lead to a paradigmatic change in the P2X 7R field and/or raise new questions about this issue.

A nomenclature for ligand-gated ion channels

The ligand-gated ion channels that participate in fast synaptic transmission comprise the nicotinic acetylcholine, 5-hydroxytryptamine3 (5-HT3), γ-aminobutyric acidA (GABAA), glycine, ionotropic glutamate and P2X receptor families. A consistent and systematic nomenclature for the individual subunits that comprise these receptors and the receptors that result from their co-assembly is highly desirable. There is also a need to develop criteria that aid in deciding which of the vast number of heteromeric combinations of subunits that can be assembled in heterologous expression systems in vitro, are known, or likely, to exist as functional receptors in vivo. The aim of this short article is to summarize the progress being made by the nomenclature committee of IUPHAR (NC-IUPHAR) in formulating recommendations that attempt to address these issues.

Trace metals in the brain: allosteric modulators of ligand-gated receptor channels, the case of ATP-gated P2X receptors

Zinc and copper are indispensable trace metals for life with a recognized role as catalysts in enzyme actions. We now review evidence supporting the role of trace metals as novel allosteric modulators of ionotropic receptors: a new and fundamental physiological role for zinc and copper in neuronal and brain excitability. The review is focussed on ionotropic receptor channels including nucleotide receptors, in particular the P2X receptor family. Since zinc and copper are stored within synaptic vesicles in selected brain regions, and released to the synaptic cleft upon electrical nerve ending depolarization, it is plausible that zinc and copper reach concentrations in the synapse that profoundly affect ligand-gated ionic channels, including the ATP-gated currents of P2X receptors. The identification of key P2X receptor amino acids that act as ligands for trace metal coordination, carves the structural determinants underlying the allosteric nature of the trace metal modulation. The recognition that the identified key residues such as histidines, aspartic and glutamic acids or cysteines in the extracellular domain are different for each P2X receptor subtype and may be different for each metal, highlights the notion that each P2X receptor subtype evolved independent strategies for metal coordination, which form upon the proper three-dimensional folding of the receptor channels. The understanding of the molecular mechanism of allosteric modulation of ligand-operated ionic channels by trace metals is a new contribution to metallo-neurobiology.

Go It Alone No More—P2X7 Joins the Society of Heteromeric ATP-Gated Receptor Channels: Fig. 1.

P2X receptors (P2XR) function as ATP-gated nonselective ion channels permeable to Na+, K+, and Ca2+, and they are expressed in a wide range of excitable, epithelial/endothelial, and immune effector cell types. The channels are trimeric complexes composed of protein subunits encoded by seven different P2XR genes expressed in mammalian and other vertebrate genomes. Current genetic, biochemical, and/or physiological evidence indicates that the extended family of functional P2X receptors includes six homomeric channels composed of P2X1, P2X2, P2X3, P2X4, P2X5, or P2X7 subunits and six heteromeric channels that involve subunit pairings of P2X1/P2X2, P2X1/P2X4, P2X1/P2X5, P2X2/P2X3, P2X2/P2X6, or P2X4/P2X6. Thus, all P2XR subtypes--with the salient exception of P2X7R--have previously been implicated in the assembly of heteromeric ATP-gated ion channels that can comprise unique pharmacological targets in different tissues. The assumed "go-it alone" function of the P2X7R has important implications because agents that target this particular receptor have been proposed as useful therapeutics in various autoinflammatory diseases or amelioration of inflammatory pain. However, this assumption and the interpretations based on it now require reevaluation in light of a new report in this issue of Molecular Pharmacology (p. 1447) that provides convincing biochemical and electrophysiological evidence for the existence of P2X4/P2X7 heteromeric receptors.

Acidic Amino Acids Impart Enhanced Ca2+ Permeability and Flux in Two Members of the ATP-gated P2X Receptor Family

P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca2+ permeability of the P2X2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca2+ currents (Pf%) of the rat and human P2X1 and P2X4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X1 receptor but had no effect on the lower Pf% of the P2X2 receptor, suggesting that ionized side chains regulate the Ca2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X1 and P2X4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X2 receptor. Taken together, the data suggest that COO− side chains provide an electrostatic force that interacts with Ca2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X1 and P2X4 receptors is conserved in the P2X3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His45 increased Pf% of the P2X3 channel, suggesting that this positive charge masks the facilitation of Ca2+ flux by the neighboring Glu46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca2+ permeability and flux of some P2X receptors.

Differential role of extracellular histidines in copper, zinc, magnesium and proton modulation of the P2X7 purinergic receptor

The P2X7 receptor is a non-selective cationic channel activated by extracellular ATP, belonging to the P2X receptor family. To assess the role of extracellular histidines on the allosteric modulation of the rat P2X7 receptor by divalent metals (copper, zinc and magnesium) and protons, these amino acid residues were singly substituted for corresponding alanines. Wild-type and mutated receptors were injected to Xenopus laevis oocytes; metal-related effects were evaluated by the two-electrode voltage-clamp technique. Copper inhibited the ATP-gated currents with a median inhibitory concentration of 4.4 +/- 1.0 micromol/L. The inhibition was non-competitive and time-dependent; copper was 60-fold more potent than zinc. The mutant H267A, resulted in a copper resistant receptor; mutants H201A and H130A were less sensitive to copper inhibition (p < 0.05). The rest of the mutants examined, H62A, H85A, and H219A, conserved the copper-induced inhibition. Only mutants H267A and H219A were less sensitive to the modulator action of zinc. Moreover, the magnesium-induced inhibition was abolished exclusively on the H130A and H201A mutants, suggesting that this metal may act at a novel cationic modulator site. Media acidification inhibited the ATP-gated current 87 +/- 3%; out of the six mutants examined, only H130A was significantly less sensitive to the change in pH, suggesting that His-130 could be involved as a pH sensor. In conclusion, while His-267 is critically involved in the copper or zinc allosteric modulation, the magnesium inhibitory effects is related to His-130 and His-201, His-130 is involved in proton sensing, highlighting the role of defined extracellular histidines in rat P2X7 receptor allosteric modulation.

The role of P2X7 in pain and inflammation

The P2X7 purinoceptor is unique amongst the P2X receptor family in that its activation is able to stimulate the release of mature, biologically active interleukin-1β (IL-1β), as well as a variety of other proinflammatory cytokines. Coupled with the predominate localisation of this receptor to immunocytes of haemopoetic origin, this receptor is an obvious candidate to play a major and pivotal role in processes of pain and inflammation. Using genetically modified animals that lack the P2X7 receptor, several investigators have shown that these mice do indeed demonstrate a blunted inflammatory response, and fail to develop pain following both inflammatory and neuropathic insult. These animals also show altered cytokine production in response to inflammatory stimulus, which is far broader than merely modulation of IL-1β release. In this short article, we review the role of the P2X7 receptor in modulating the release of cytokines and other mediators, and discuss the findings made from P2X7 receptor-deficient animals. As well as highlighting outstanding questions regarding this intriguing receptor, we also speculate as to the potential therapeutic benefit of P2X7 receptor modulation.

PIP2­ Regulates the Ionic Current of P2X Receptors and P2X­7 Receptor-Mediated Cell Death

P2X7 receptors are non-selective cation channels gated by extracellular ATP. Prolonged stimulation of the P2X7 receptor leads to an increase in cell permeability, cytokine release and apoptosis/necrosis. Application of PIP2 to inside-out patches strongly activated all homomeric members of the P2X receptor family, including P2X7 channels. Blockade of PIP2 re-synthesis or induction of PIP2 hydrolysis diminished the ATP-gated P2X7 currents. Several positively charged residues in the proximal C-terminus of P2X7 were found to be important for PIP2 interactions, as mutation of these sites reduced the apparent affinity for PIP2 and enhanced current inhibition by PIP2 depletion. In addition, we demonstrated the dependence on the interaction of the receptor with PIP2 of ATP-mediated cell death in HEK cells stably transfected with P2X7, in primary T cells and in macrophages. These results identify PIP2 as a critical regulator of the function of the extracellular ligand-gated P2X receptor/channels and provide a novel way to control ATP-mediated cell death.

Vesicular release of ATP at central synapses

Adenosine triphosphate (ATP) acts as a fast excitatory transmitter in several regions of the central nervous system (CNS) including the medial habenula, dorsal horn, locus coeruleus, hippocampus, and somatosensory cortex. Postsynaptic actions of ATP are mediated through an extended family of P2X receptors, widely expressed throughout the CNS. ATP is released via several pathways, including exocytosis from presynaptic terminals and diffusion through large transmembrane pores (e.g., hemichannels, P2X(7) receptors, or volume-sensitive chloride channels) expressed in astroglial membranes. In presynaptic terminals, ATP is accumulated and stored in the synaptic vesicles. In different presynaptic terminals, these vesicles may contain ATP only or ATP and another neurotransmitter [e.g., gamma-amino-butyric acid (GABA) or glutamate]; in the latter case, two transmitters can be coreleased. Here, we discuss the mechanisms of vesicular release of ATP in the CNS and present our own data, which indicate that in central neuronal terminals, ATP is primarily stored and released from distinct pool of vesicles; the release of ATP is not synchronized either with GABA or with glutamate.

Visualization of the trimeric P2X 2 receptor with a crown-capped extracellular domain

The P2X 2 purinergic receptor permeates cationic ions in response to stimulation by ATP and mediates fast synaptic transmission. Here, we purified the P2X 2 receptor using baculovirus-Sf9 cell expression system and observed its structure using electron microscopy. The FLAG-tagged P2X 2 receptor, which has intact ion channel function, was purified to be a single peak by affinity purification and gel filtration chromatography. It was confirmed to be a trimer by introducing cross-linking. Negatively stained P2X 2 protein images were homogeneous and picked up by automated pick-up programs, aligned, and classified using the modified growing neural gas network method. Similarly oriented projections were averaged to decrease the signal-to-noise ratio. These images demonstrate an inverted three-sided pyramid with the dimensions of 215 Å in height and 200 Å in side length. It is composed of a high-density trunk and a stain-permeable swollen extracellular domain of a crown-shaped structure. The internal cavities and constituent segments were clearly demonstrated in both the raw images and the averaged images. The threefold symmetrical top view demonstrates the first visual evidence of the trimeric composition of the P2X receptor family.

“Host Tissue Damage” Signal ATP Promotes Non-directional Migration and Negatively Regulates Toll-like Receptor Signaling in Human Monocytes

The activation of Toll-like receptors (TLRs) by lipopolysaccharide or other ligands evokes a proinflammatory immune response, which is not only capable of clearing invading pathogens but can also inflict damage to host tissues. It is therefore important to prevent an overshoot of the TLR-induced response where necessary, and here we show that extracellular ATP is capable of doing this in human monocytes. Using reverse transcription-PCR, we showed that monocytes express P2Y(1), P2Y(2), P2Y(4), P2Y(11), and P2Y(13) receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca(2+) signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca(2+), which corresponded to internal Ca(2+) release followed by activation of store-operated Ca(2+) entry. The evoked Ca(2+) signals stimulated Ca(2+)-activated K(+) channels, producing transient membrane hyperpolarization. In addition, ATP promoted cytoskeleton reorganization and cell migration; however, unlike chemoattractants, the migration was non-directional and further analysis showed that ATP did not activate Akt, essential for sensing gradients. When TLR2, TLR4, or TLR2/6 were stimulated with their respective ligands, ATPgammaS profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-alpha and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATPgammaS) strongly increased cAMP levels, and, moreover, the TLR-response was inhibited by forskolin, whereas UTP neither increased cAMP nor inhibited the TLR-response. Thus, our data suggest that ATP promotes non-directional migration and, importantly, acts as a "host tissue damage" signal via the G(s) protein-coupled P2Y(11) receptor and increased cAMP to negatively regulate TLR signaling.

A Role for Mitogen-activated Protein KinaseErk1/2 Activation and Non-selective Pore Formation in P2X7 Receptor-mediated Thymocyte Death

Extracellular ATP (ATPe) binds to P2X7 receptors (P2X7R) expressed on the surface of cells of hematopoietic lineage, including murine thymocytes. Activation of P2X7R by ATPe results in the opening of cation-specific channels, and prolonged ATPe exposure leads to the formation of non-selective pores enabling transmembrane passage of solutes up to 900 Da. In the presence of ATPe, P2X7R-mediated thymocyte death is due primarily to necrosis/lysis and not apoptosis, as measured by the release of lactate dehydrogenase indicative of a loss of plasma membrane integrity. The present study is focused on the identification of P2X7R signaling mediators in ATP-induced thymocyte necrosis/lysis. Thus, extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation was found to be required for cell lysis, and both events were independent of ATP-induced calcium influx. P2X7R-dependent thymocyte death involved the chronological activation of Src family tyrosine kinase(s), phosphatidylinositol 3-kinase, the mitogen-activated protein (MAP) kinase(Erk1/2) module, and the proteasome. Although independent of this signaling cascade, non-selective pore formation may modulate ATP-mediated thymocyte death. These results therefore suggest a role for both activation of MAP kinase(Erk1/2) and non-selective pore opening in P2X7R-induced thymocyte death.

Overview of the P2 Receptors

The release of nucleotides in extracellular fluids can result from cell necrosis, exocytosis of secretory granules (such as platelet dense granules), or efflux through membrane channels. In addition, recent evidence suggests that vesicular trafficking is an important pathway of nucleotide release. Once in the extracellular fluids, they are rapidly degraded by ectonucleotidases, such as CD39, that play a key role in neutralizing the platelet aggregatory action of adenosine diphosphate (ADP), and act on two families of receptors: the ionotropic P2X receptors and the G-protein-coupled P2Y receptors. The family of P2X receptors encompasses seven genes. Currently, there are eight genuine P2Y receptors that can be subdivided into two structurally distinct subfamilies. Whereas P2X receptors are receptors of ATP, the different P2Y receptors are activated by distinct nucleotides, diphosphates or triphosphates, or purines or pyrimidines, some of them being conjugated to sugars. The study of knockout mice has demonstrated that P2X receptors play important roles in the neurogenic control of smooth muscle contraction, in pain and visceral perception, and in macrophage functions. The phenotype of P2Y null mice so far is more restricted: inhibition of platelet aggregation to ADP and increased bleeding time in P2Y (1)(-/-) and P2Y (12)(-/-) mice and lack of epithelial responsiveness to nucleotides in airways (P2Y (2)(-/-)) and intestine (P2Y (4)(0/-)).

P2X1 receptors and the endothelium

Adenosine triphosphate (ATP) is now established as a principle vaso-active mediator in the vasculature. Its actions on arteries are complex, and are mediated by the P2X and P2Y receptor families. It is generally accepted that ATP induces a bi-phasic response in arteries, inducing contraction via the P2X and P2Y receptors on the smooth muscle cells, and vasodilation via the actions of P2Y receptors located on the endothelium. However, a number of recent studies have placed P2X1 receptors on the endothelium of some arteries. The use of a specific P2X1 receptor ligand, alpha, beta methylene ATP has demonstrated that P2X1 receptors also have a bi-functional role. The actions of ATP on P2X1 receptors is therefore dependant on its location, inducing contraction when located on the smooth muscle cells, and dilation when expressed on the endothelium, comparable to that of P2Y receptors.

The cytokine IL-1beta transiently enhances P2X7 receptor expression and function in human astrocytes.

Extracellular nucleotide di- and triphosphates such as ATP and ADP mediate their effects through purinergic P2 receptors belonging to either the metabotropic P2Y or the ionotropic P2X receptor family. The P2X7R is a unique member of the P2X family, which forms a pore in response to ligand stimulation, regulating cell permeability, cytokine release, and/or apoptosis. This receptor is also unique in that its affinity for the ligand benzoyl-benzoyl ATP (BzATP) is at least 10-fold greater than that of ATP. Primary human fetal astrocytes in culture express low-levels of P2X7R mRNA and protein, and BzATP induces only a slight influx in intracellular calcium [Ca2+]i, with little demonstrable effect on gene expression or pore formation in these cells. We now show that, following treatment with the proinflammatory cytokine IL-1beta, BzATP induces a robust rise in [Ca2+]i with agonist and antagonist profiles indicative of the P2X7R. IL-1beta also induced the formation of membrane pores as evidenced by the uptake of YO-PRO-1 (375 Da). Quantitative real-time PCR demonstrated transient upregulation of P2X7R mRNA in IL-1beta-treated cells, while FACS analysis indicated a similar upregulation of P2X7R protein at the cell membrane. In multiple sclerosis lesions, immunoreactivity for the P2X7R was demonstrated on reactive astrocytes in autopsy brain tissues. In turn, P2X7R stimulation increased the production of IL-1-induced nitric oxide synthase activity by astrocytes in culture. These studies suggest that signaling via the P2X7R may modulate the astrocytic response to inflammation in the human central nervous system.

The First Transmembrane Domain of the P2X Receptor Subunit Participates in the Agonist-induced Gating of the Channel

Based on pharmacological properties, the P2X receptor family can be subdivided into those homo-oligomers that are sensitive to the ATP analog alphabeta-methylene ATP(alphabetameATP) (P2X(1) and P2X(3)) and those that are not (P2X(2), P2X(4), P2X(5), P2X(6), and P2X(7)). We exploited this dichotomy through the construction of chimeric receptors and site-directed mutagenesis in order to identify domains responsible for these differences in the abilities of extracellular agonists to gate P2X receptors. Replacement of the extracellular domain of the alphabetameATP-sensitive rat P2X(1) subunit with that of the alphabetameATP-insensitive rat P2X(2) subunit resulted in a receptor that was still alphabetameATP-sensitive, suggesting a non-extracellular domain was responsible for the differential gating of P2X receptors by various agonists. Replacement of the first transmembrane domain of the rat P2X(2) subunit with one from an alphabetameATP-sensitive subunit (either rat P2X(1) or P2X(3) subunit) converted the resulting chimera to alphabetameATP sensitivity. This conversion did not occur when the first transmembrane domain came from a non-alphabetameATP-sensitive subunit. Site-directed mutagenesis indicated that the C-terminal portion of the first transmembrane domain was important in determining the agonist selectivity of channel gating for these chimeras. These results suggest that the first transmembrane domain plays an important role in the agonist operation of the P2X receptor.

Extracellular ATP induces a distorted maturation of dendritic cells and inhibits their capacity to initiate Th1 responses.

Dendritic cells (DCs) express functional purinergic receptors, but the effects of purine nucleotides on DC functions have been marginally investigated. In this study, we report on the ability of micromolar concentrations of ATP to affect the maturation and Ag-presenting function of monocyte-derived DCs in vitro. Chronic stimulation (24 h) of DCs with low, noncytotoxic ATP doses increased membrane expression of CD54, CD80, CD86, and CD83, slightly reduced the endocytic activity of DCs, and augmented their capacity to promote proliferation of allogeneic naive T lymphocytes. Moreover, ATP enhanced LPS- and soluble CD40 ligand-induced CD54, CD86, and CD83 expression. On the other hand, ATP markedly and dose-dependently inhibited LPS- and soluble CD40 ligand-dependent production of IL-1alpha, IL-1beta, TNF-alpha, IL-6, and IL-12, whereas IL-1 receptor antagonist and IL-10 production was not affected. As a result, T cell lines generated from allogeneic naive CD45RA(+) T cells primed with DCs matured in the presence of ATP produced lower amounts of IFN-gamma and higher levels of IL-4, IL-5, and IL-10 compared with T cell lines obtained with LPS-stimulated DCs. ATP inhibition of TNF-alpha and IL-12 production by mature DCs was not mediated by PGs or elevation of intracellular cAMP and did not require ATP degradation. The inability of UTP and the similar potency of ADP to reproduce ATP effects indicated that ATP could function through the P2X receptor family. These results suggest that extracellular ATP may serve as an important regulatory signal to dampen IL-12 production by DCs and thus prevent exaggerated and harmful immune responses.

Procedures to characterize and study P2Z/P2X7 purinoceptor: flow cytometry as a promising practical, reliable tool

The expression of P2Z/P2X7 purinoceptor in different cell types is well established. This receptor is a member of the ionotropic P2X receptor family, which is composed by seven cloned receptor subtypes (P2X1 - P2X7). Interestingly, the P2Z/P2X7 has a unique feature of being linked to a non-selective pore which allows the passage of molecules up to 900 Da depending on the cell type. Early studies of P2Z/P2X7 purinoceptor were exclusively based on classical pharmacological studies but the recent tools of molecular biology have enriched the analysis of the receptor expression. The majority of assays and techniques chosen so far to study the expression of P2Z/P2X7 receptor explore directly or indirectly the effects of the opening of P2Z/P2X7 linked pore. In this review we describe the main techniques used to study the expression and functionality of P2Z/P2X7 receptor. Additionally, the increasing need and importance of a multifunctional analysis of P2Z/P2X7 expression based on flow cytometry technology is discussed, as well as the adoption of a more complete analysis of P2Z/P2X7 expression involving different techniques.

Expression of two ATP-gated ion channels, P2X5 and P2X6, in developing chick skeletal muscle.

Physiological and pharmacological studies have shown that ATP has potent effects on developing chick skeletal muscle. These effects have previously been shown to be developmentally regulated, and the responses were characteristic of activation of the P2X ligand-gated ion-channel family of ATP receptors. Here, using immunohistochemistry, we describe the expression patterns of two members of the P2X receptor family, P2X5 and P2X6, during development of skeletal muscle in the chick embryo. These receptors were first expressed at early stages of skeletal muscle development, and expression disappeared immediately before the stage at which fusion of myoblasts to form myotubes occurs. P2X5 was also demonstrated in nerves supplying developing skeletal muscle, in some dorsal root ganglion cells, and in dorsal and ventral spinal cord. No expression of the other five members of the P2X family were demonstrated in developing skeletal muscle.