High potency zinc modulation of human P2X2 receptors and low potency zinc modulation of rat P2X2 receptors share a common molecular mechanism

Human P2X2 receptors (hP2X2) are strongly inhibited by zinc over the range of 2–100 μm, whereas rat P2X2 receptors (rP2X2) are strongly potentiated over the same range, and then inhibited by zinc over 100 μm. However, the biological role of zinc modulation is unknown in either species. To identify candidate regions controlling zinc inhibition in hP2X2 a homology model based on the crystal structure of zebrafish P2X4.1 was made. In this model, His-204 and His-209 of one subunit were near His-330 of the adjacent subunit. Cross-linking studies confirmed that these residues are within 8 Å of each other. Simultaneous mutation of these three histidines to alanines decreased the zinc potency of hP2X2 nearly 100-fold. In rP2X2, one of these histidines is replaced by a lysine, and in a background in which zinc potentiation was eliminated, mutation of Lys-197 to histidine converted rP2X2 from low potency to high potency inhibition. We explored whether the zinc-binding site lies within the vestibules running down the central axis of the receptor. Elimination of all negatively charged residues from the upper vestibule had no effect on zinc inhibition. In contrast, mutation of several residues in the hP2X2 middle vestibule resulted in dramatic changes in the potency of zinc inhibition. In particular, the zinc potency of P206C could be reversibly shifted from extremely high (∼10 nm) to very low (>100 μm) by binding and unbinding MTSET. These results suggest that the cluster of histidines at the subunit interface controls access of zinc to its binding site.     

Agonist versus antagonist action of ATP at the P2Y4 receptor is determined by the second extracellular loop

UTP is a potent full agonist at both the human P2Y(4) (hP2Y(4)) and rat P2Y(4) (rP2Y(4)) receptor. In contrast, ATP is a potent full agonist at the rP2Y(4) receptor but is a similarly potent competitive antagonist at the hP2Y(4) receptor. To delineate the structural determinants of agonism versus antagonism in these species homologues, we expressed a series of human/rat P2Y(4) receptor chimeras in 1321N1 human astrocytoma cells and assessed the capacity of ATP and UTP to mobilize intracellular Ca(2+). Replacement of the NH(2) terminus of the hP2Y(4) receptor with the corresponding region of the rP2Y(4) receptor resulted in a receptor that was activated weakly by ATP, whereas replacement of the second extracellular loop (EL2) of the hP2Y(4) receptor with that of the rP2Y(4) receptor yielded a chimeric receptor that was activated fully by UTP and near fully by ATP, albeit with lower potencies than those observed at the rP2Y(4) receptor. These potencies were increased, and ATP was converted to a full agonist by replacing both the NH(2) terminus and EL2 in the hP2Y(4) receptor with the corresponding regions from the rP2Y(4) receptor. Mutational analysis of the five divergent amino acids in EL2 between the two receptors revealed that three amino acids, Asn-177, Ile-183, and Leu-190, contribute to the capacity of EL2 to impart ATP agonism. Taken together, these results suggest that the second extracellular loop and the NH(2) terminus form a functional motif that plays a key role in determining whether ATP functions as an agonist or antagonist at mammalian P2Y(4) receptors.     

Establishment of an assay for P2X7 receptor-mediated cell death

The P2X7 receptor, an ATP-gated cation channel, induces cell death in immune cells and is involved in neurodegenerative diseases. Although the receptor plays various roles in these diseases, the cellular mechanisms involved are poorly understood and antagonists are limited. Here, the development of a cell-based assay for human P2X7 receptor is reported. We established permanent lines of HEK 293 cells expressing a high level of hP2X7 receptor. Functional activity of the hP2X7 receptor was confirmed by whole-cell patch recording of ATP-induced ion currents. Prolonged exposure to ATP resulted in death of the hP2X7-expressing HEK 293 cells and this cell death could be quantified. Two known P2X7 antagonists, PPADS and KN-62, blocked ATP-induced death in a concentration-dependent manner. Thus, this assay can be used to screen for new antagonists of hP2X7 receptors.     

A His-155 to Tyr polymorphism confers gain-of-function to the human P2X7 receptor of human leukemic lymphocytes

The P2X(7)R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X(7)R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X(7)R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X(7)R function was monitored by measuring ATP-induced Ca(2+) influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X(7) allelic variant. Ca(2+) influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca(2+) flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X(7)R confirmed an increased ATP-dependent activation of the P2X(7) 489T mutant with respect to the wild type receptor, as assessed by both by [Ca(2+)](i) influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X(7)R.     

Gain-of-function of P2X7 receptor gene variants in multiple sclerosis

We have previously shown that P2X7 receptor blockade prevents ATP excitotoxicity in oligodendrocytes and ameliorates chronic experimental autoimmune encephalomyelitis. Here, we have explored the putative association of functionally relevant single nucleotide polymorphisms of the P2X7 receptor gene with multiple sclerosis. We found that T allele of rs17525809 polymorphism, which yields an Ala-76 to Val change in the extracellular domain, is more frequent in multiple sclerosis patients than in controls. Importantly, P2X7 variants with Val show a gain-of-function consisting in higher calcium permeability, larger electrophysiological responses and higher ethidium uptake, and enhance the effect of the also gain-of-function His-155 to Tyr substitution (rs208294) in the haplotype formed by these two variants. These findings may contribute to define the genetic background predisposing for multiple sclerosis and its pathophysiology.     

P2Y<Subscript>1</Subscript> receptor modulation of endogenous ion channel function in <Emphasis Type="Italic">Xenopus</Emphasis> oocytes: Involvement of transmembrane domains

Agonist activation of the hP2Y₁ receptor expressed in Xenopus oocytes stimulated an endogenous voltage-gated ion channel, previously identified as the transient inward (T_in) channel. When human P2Y₁ (hP2Y₁) and skate P2Y (sP2Y) receptors were expressed in Xenopus oocytes, time-to-peak values (a measure of the response to membrane hyperpolarization) of the T_in channel were significantly reduced compared to oocytes expressing the hB₁-bradykinin receptor or the rat M₁-muscarinic (rM₁) receptor. Differences in activation were also observed in the T_in currents elicited by various P2Y receptor subtypes. The time-to-peak values of the T_in channel in oocytes expressing the hP2Y₄, hP2Y₁₁, or hB₁-bradykinin receptors were similar, whereas the channel had significantly shorter time-to-peak values in oocytes expressing either the hP2Y₁ or sP2Y receptor. Amino acid substitutions at His-132, located in the third transmembrane domain (TM3) of the hP2Y₁ receptor, delayed the onset of channel opening, but not the kinetics of the activation process. In addition, Zn²⁺ sensitivity was also dependent on the subtype of P2Y receptor expressed. Replacement of His-132 in the hP2Y₁ receptor with either Ala or Phe increased Zn²⁺ sensitivity of the T_in current. In contrast, truncation of the C-terminal region of the hP2Y₁ receptor had no affect on activation or Zn²⁺ sensitivity of the T_in channel. These results suggested that TM3 in the hP2Y₁ receptor was involved in modulating ion channel function and blocker pharmacology of the T_in channel.     

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium⁺ uptake into KG-l cells suspended in sucrose medium (EC₅₀ of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium⁺ uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC₅₀ of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC₅₀ of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium⁺ uptake into KG-1 cells. ATP-induced ethidium⁺ uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium⁺ uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1²⁺ and propidium²⁺ uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells.     

Autoactivation of type-1 parathyroid hormone receptors containing a tethered ligand

Interactions between the N-terminal residues of parathyroid hormone (PTH) and the region of the PTH receptor containing the extracellular loops and transmembrane domains are thought to be critical for receptor activation. We evaluated this hypothesis by replacing the large N-terminal extracellular domain of the human type 1 PTH receptor (hP1Rc-WT) with residues 1-9 of PTH (AVSEIQLMH) using a tetraglycine linker between His-9 of the ligand and Glu-182 of the receptor near the extracellular terminus of transmembrane domain-1. Expression of this construct, hP1Rc-Tether(1-9), in COS-7 cells resulted in basal cAMP levels that were 10-fold higher than those seen in control cells transfected with hP1Rc-WT. Extending the ligand sequence to include Asn-10 and the activity-enhancing substitution of Leu-11 --> Arg yielded hP1Rc-[Arg(11)]Tether(1-11), for which we observed basal cAMP levels that were 50-fold higher than those seen with P1Rc-WT. An alanine-scan analysis of hP1Rc-[Arg(11)]Tether(1-11) revealed that Gln-6 and His-9 were not critical for autoactivation, whereas Val-2, Ile-5, and Met-8 were. The data show that tethered PTH/PTH receptors can autoactivate. Analysis of the structure-activity relationships in these tethered receptor constructs can provide new information concerning how the N-terminal residues of PTH interact with the extracellular loops and transmembrane regions of the PTH-1 receptor, particularly in regard to receptor activation.     

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.     

Structural basis for the functional properties of the P2X7 receptor for extracellular ATP

The P2X7 receptor, originally known as the P2Z receptor due to its distinctive functional properties, has a structure characteristic of the ATP-gated ion channel P2X receptor family. The P2X7 receptor is an important mediator of ATP-induced purinergic signalling and is involved the pathogenesis of numerous conditions as well as in the regulation of diverse physiological functions. Functional characterisations, in conjunction with site-directed mutagenesis, molecular modelling, and, recently, structural determination, have provided significant insights into the structure–function relationships of the P2X7 receptor. This review discusses the current understanding of the structural basis for the functional properties of the P2X7 receptor.     

Characteristics of P2X7 receptors from human B lymphocytes expressed in Xenopus oocytes

Human B lymphocytes express an ATP-gated ion channel (P2Z receptor), which shares similarities with the recently identified P2X7 receptor. Using gene specific primers, we have now isolated P2X7 cDNA from the total RNA of human B lymphocytes. This hP2X7 receptor subtype was expressed in Xenopus oocytes and electrophysiologically characterized. The hP2X7 receptor is similar to, but does not completely match, P2Z of human B cells. The hP2X7 receptors resemble the P2Z receptors with regard to the ATP concentration of half maximal activation, reproducibility, permeation characteristics and lack of desensitization of the ATP-evoked currents. However, in contrast to the native lymphocytic P2Z receptor, the time course of activation of hP2X7 displayed an additional linearly increasing current component. Furthermore, a second, small and slowly deactivating current component exists only in hP2X7 expressed in oocytes. The activation and deactivation kinetics as well as permeation characteristics of hP2X7 are different from rat P2X7 recently expressed in oocytes. Unlike in mammalian cells, hP2X7 expressed in Xenopus oocytes is not sufficient to induce large non-selective pores.     

Influence of extracellular monovalent cations on pore and gating properties of P2X7 receptor-operated single-channel currents

Using the patch-clamp method, we studied the influence of external alkali and organic monovalent cations on the single-channel properties of the adenosine triphosphate (ATP)-activated recombinant human P2X(7) receptor. The slope conductance of the hP2X(7) channel decreased and the reversal potential was shifted to more negative values as the ionic diameter of the organic test cations increased. From the relationship between single-channel conductance and the dimensions of the inward current carrier, the narrowest portion of the pore was estimated to have a mean diameter of approximately 8.5 A. Single-channel kinetics and permeation properties remained unchanged during receptor activation by up to 1 mM ATP(4-) for >1 min, arguing against a molecular correlate of pore dilation at the single P2X(7) channel level. Substitution of extracellular Na(+) by any other alkali or organic cation drastically increased the open probability of the channels by prolonging the mean open time. This effect seems to be mediated allosterically through an extracellular voltage-dependent Na(+) binding site with a K(d) of approximately 5 mM Na(+) at a membrane potential of -120 mV. The modulation of the ATP-induced hP2X(7) receptor gating by extracellular Na(+) could be well described by altering the rate constant from the open to the neighboring closed state in a C-C-C-O kinetic receptor model. We suggest that P2X(7) receptor-induced depolarization and associated K(+)-efflux may reduce Na(+) occupancy of the regulatory Na(+) binding site and thus increase the efficacy of ATP(4-) in a feed-forward manner in P2X(7) receptor-expressing cells.     

Promotion of cathepsin L activity in newt spermatogonial apoptosis induced by prolactin

Residues considered essential for ATP binding to the human P2X(7) receptor (hP2X(7)R) were investigated. HEK293 cells or Xenopus oocytes were transfected with wild-type or site-directed mutants of hP2X(7)R constructs and channel/pore activity measured in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). Barium uptake and ethidium influx into HEK293 cells were abolished in cells expressing K193A and K311A mutants, and were partially reduced in cells expressing mutant P210A. K193A and K311A mutations also completely abolished responses to ATP and BzATP in Xenopus oocytes as measured by electrophysiology. These results indicate that K193 and K311 are essential residues in ATP binding in the hP2X(7)R.     

An Ile-568 to Asn polymorphism prevents normal trafficking and function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X(7) receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into various lymphocyte subsets and was decreased to values of approximately 25% of normal. The expression of the P2X(7) receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X(7) carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-gamma up-regulated P2X(7) function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X(7) receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.     

The effect of anions on the human P2X7 receptor

P2X7 receptors (P2X7Rs) are nonselective cation channels that are opened by the binding of extracellular ATP and are involved in the modulation of epithelial secretion, inflammation and nociception. Here, we investigated the effect of extracellular anions on channel gating and permeation of human P2X7Rs (hP2X7Rs) expressed in Xenopus laevis oocytes. Two-microelectrode voltage-clamp recordings showed that ATP-induced hP2X7R-mediated currents increased when extracellular chloride was substituted by the organic anions glutamate or aspartate and decreased when chloride was replaced by the inorganic anions nitrate, sulfate or iodide. ATP concentration-response comparisons revealed that substitution of chloride by glutamate decreased agonist efficacy, while substitution by iodide increased agonist efficacy at high ATP concentrations. Meanwhile, the ATP potency remained unchanged. Activation of the hP2X7R at low ATP concentrations via the high-affinity ATP effector site was not affected by the replacement of chloride by glutamate or iodide. To analyze the anion effect on the hP2X7R at the single-molecule level, we performed single-channel current measurements using the patch-clamp technique in the outside-out configuration. Chloride substitution did not affect the single-channel conductance, but the probability that the P2X7R channel was open increased when chloride was replaced by glutamate and decreased when chloride was replaced by iodide. This effect was due to an influence of the anions on the mean closed times of the hP2X7R channel. We conclude that hP2X7R channels are not anion-permeable in physiological Na+-based media and that external anions allosterically affect ion channel opening in the fully ATP4-liganded P2X7R through an extracellular anion binding site.     

Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating

P2X3 receptors (P2X3R) are trimeric ATP-gated cation channels involved in sensory neurotransmission and inflammatory pain. We used homology modeling and molecular dynamic simulations of the hP2X3R to identify inter-subunit interactions of residues that are instrumental to elucidate conformational changes associated with gating of the hPX3R. We identified an ionic interaction between E112 and R198 of the head domain and dorsal fin domain, respectively, and E57 and T263 of the lower body domains of adjacent subunits and detected a marked rearrangement of these domains during gating of the hP3X3R. Double-mutant cycle analysis of the inter-subunit residue pairs E112/R198 and E57/T263 revealed significant interaction-free energies. Disulfide locking of the hP2X3R E112C/R198C or the E57C/T263C double cysteine mutants markedly reduced the ATP-induced current responses. The decreased current amplitude following inter-subunit disulfide cross-linking indicates that disulfide locking of the head and dorsal fin domains or at the level of the lower body domains of the hP2X3R prevents the gating-induced conformational rearrangement of the subunits with respect to each other. The distinct reorganization of the subunit interfaces during gating of the hP2X3R is generally consistent with the gating mechanism of other P2XRs. Charge-reversal mutagenesis and methanethiosulfonate (MTS)-modification of substituted cysteines demonstrated that E112 and R198 interact electrostatically. Both disulfide locking and salt bridge breaking of the E112/R198 interaction reduced the hP2X3R function. We conclude that the inter-subunit salt bridge between E112 and R198 of the head and dorsal fin domains, respectively, serves to control the mobility of these domains during agonist-activation of the hP2X3R.     

Identification and characterization of a novel variant of the human P2X<Subscript>7</Subscript> receptor resulting in gain of function

The P2X₇ receptor exhibits significant allelic polymorphism in humans, with both loss and gain of function variants potentially impacting on a variety of infectious and inflammatory disorders. At least five loss-of-function polymorphisms (G150R, R307Q, T357S, E496A, and I568N) and two gain-of-function polymorphisms (H155Y and Q460R) have been identified and characterized to date. In this study, we used RT-PCR cloning to isolate and characterize P2X₇ cDNA clones from human PBMCs and THP-1 cells. A previously unreported variant with substitutions of V80M and A166G was identified. When expressed in HEK293 cells, this variant exhibited heightened sensitivity to the P2X₇ agonist (BzATP) relative to the most frequent allele, as shown by pore formation measured by fluorescent dye uptake into cells. Mutational analyses showed that A166G alteration was critical for the gain-of-function change, while V80M was not. Full-length variants with multiple previously identified nonsynonymous SNPs (H155Y, H270R, A348T, and E496A) were also identified. Distinct functional phenotypes of the P2X₇ variants or mutants constructed with multiple polymorphisms were observed. Gain-of-function variations (A166G or H155Y) could not rescue the loss-of-function E496A polymorphism. Synergistic effects of the gain-of-function variations were also observed. We also identified the A348T alteration as a weak gain-of-function variant. Thus, these results identify the new gain-of-function variant A166G and demonstrate that multiple-gene polymorphisms contribute to functional phenotypes of the human P2X₇ receptor. Furthermore, the results demonstrate that the C-terminal of the cysteine-rich domain 1 of P2X₇ is critical for regulation of P2X₇-mediated pore formation.     

A Thr357 to Ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs ATP-induced mycobacterial killing by macrophages

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.     

Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.