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1.
Oncogene ; 30(18): 2108-22, 2011 May 05.
Article in English | MEDLINE | ID: mdl-21242969

ABSTRACT

ATP-gated P2X(7) receptors (P2X(7)R) are unusual plasma membrane ion channels that have been extensively studied in immune cells. More recently, P2X(7)R have been described as potential cancer cell biomarkers. However, mechanistic links between P2X(7)R and cancer cell processes are unknown. Here, we show, in the highly aggressive human breast cancer cell line MDA-MB-435s, that P2X(7) receptor is highly expressed and fully functional. Its activation is responsible for the extension of neurite-like cellular prolongations, of the increase in cell migration by 35% and in cell invasion through extracellular matrix by 150%. The change in cancer cell morphology and the increased migration appeared to be due to the activation of Ca(2+)-activated SK3 potassium channels. The enhanced invasion through the extracellular matrix was related to the increase of mature forms of cysteine cathepsins in the extracellular medium, which was independent of SK3 channel activity and not associated with cell death. Pharmacological targeting of P2X(7)R in vivo was crucial for cell invasiveness in a zebrafish model of metastases. Our results demonstrate a novel mechanistic link between P2X(7)R functionality in cancer cells and invasiveness, a key parameter in tumour growth and in the development of metastases. They also suggest a potential therapeutic role for the newly developed P2X(7)R antagonists.


Subject(s)
Cathepsins/physiology , Neoplasm Invasiveness , Purinergic Agonists/pharmacology , Receptors, Purinergic P2X7/drug effects , Small-Conductance Calcium-Activated Potassium Channels/physiology , Cell Line, Tumor , Humans
2.
FEBS Lett ; 584(23): 4740-4, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-21073871

ABSTRACT

We investigated membrane targeting of the P2X(7) receptor (P2X(7)R) in polarized epithelial cells using immunofluorescent confocal imaging. The wild-type receptor was targeted to the basolateral membrane, independently of adaptor protein µ1B. Deletion of the majority of the intracellular C-terminus, or the last 26 residues (P570-Y595), conferred targeting of the protein to the apical membrane. Alanine substitution in the microdomain P582-Q587 caused similar apical membrane targeting without major effect on the receptor function and surface expression. Our results show basolateral membrane targeting of the P2X(7)R in epithelial cells and that the intracellular C-terminal microdomain P582-Q587 is crucial in this process.


Subject(s)
Cell Membrane/metabolism , Cell Polarity , Epithelial Cells/cytology , Intracellular Space/metabolism , Receptors, Purinergic P2X7/chemistry , Receptors, Purinergic P2X7/metabolism , Alanine , Amino Acid Sequence , Amino Acid Substitution , Animals , Dogs , Epithelial Cells/metabolism , HEK293 Cells , Humans , Molecular Sequence Data , Protein Structure, Tertiary , Protein Transport , Rats , Receptors, Purinergic P2X7/deficiency , Receptors, Purinergic P2X7/genetics , Sequence Deletion
3.
Histochem Cell Biol ; 134(1): 31-8, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20563595

ABSTRACT

It has recently been shown in epithelial cells that the ATP-gated ion channel P2X7R is in part, associated with caveolae and colocalized with caveolin-1. In the present study of the mouse heart, we show for the first time, using immunohistochemistry and cryoimmunoelectron microscopy, that P2X7R is expressed in atrial cardiomyocytes and in cardiac microvascular endothelial cells, but not in the ventricle cardiomyocytes. Furthermore, biochemical data indicate the presence of two forms of P2X7R, the classical glycosylated 80 kDa isoform and a protein with the molecular weight of 56 kDa, in both cardiomyocytes and endothelial cells of the mouse heart. The functionality of both proteins in heart cells is still unclear. In cardiac tissue homogenates derived from caveolin-1 deficient mice (cav-1(-/-)), an increase of the P2Xrx7 mRNA and P2X7R protein (80 kDa) was found, particularly in atrial samples. In addition, P2rx7(-/-) mice showed enhanced protein levels of caveolin-1 in their atrial tissues. Although the details of cellular mechanisms that underlie the relationship between caveolin-1 and P2X7R in atrial cardiomyocytes and the electrophysiological consequences of the increased P2X7R expression in atrial cells of cav-1(-/-) mice remain to be elucidated, the cardiomyopathy detectable in cav-1(-/-) mice is possibly related to a disturbed crosstalk between P2X7R and caveolin-1 in different heart cell populations.


Subject(s)
Caveolin 1/deficiency , Heart Atria/cytology , Myocytes, Cardiac/metabolism , Receptors, Purinergic P2/biosynthesis , Receptors, Purinergic P2/genetics , Animals , Blotting, Western , Female , Immunohistochemistry , Mice , RNA, Messenger/genetics , Receptors, Purinergic P2X7 , Reverse Transcriptase Polymerase Chain Reaction
4.
Br J Pharmacol ; 156(1): 135-42, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19068080

ABSTRACT

BACKGROUND AND PURPOSE: P2X(7), receptors are potently inhibited by extracellular acidification. The underlying molecular basis remains unknown. This study aimed to examine the role of extracellular histidine, lysine, aspartic acid and glutamic acid residues in the functional inhibition of rat P2X(7) receptors by acidic pH. EXPERIMENTAL APPROACH: We introduced point mutations into rat P2X(7) receptor by site-directed mutagenesis, expressed wild type (WT) and mutant receptors in human embryonic kidney (HEK293) cells and, using patch clamp recording, characterized the effects of acidic pH on BzATP [2'-3'O-(4-benzoylbenzoyl) adenosine 5'-triphosphate]-evoked ionic currents. KEY RESULTS: Reducing extracellular pH, that is, increasing extracellular proton concentrations, inhibited BzATP-evoked currents in cells expressing WT P2X(7) receptors, with IC(50) value (half-maximal antagonist or inhibitor concentration) for protons of 0.2 mumol.L(-1). The major effect of acidification was suppression of the maximal current response without altering the agonist sensitivity. five residues in the receptor extracellular domain (His(85), Lys(110), Lys(137), Asp(197) and His(219)) were mutated to alanine and current inhibition by protons assessed. Compared with WT, the H85A, H219A, K137A mutants were two- to threefold more sensitive, whereas the K110A and D197A mutants were 2.5- and 9-fold less sensitive. Double-alanine substitution of Lys(110) and Asp(197) resulted in 23-fold decreased sensitivity to inhibition by protons. Furthermore, charge neutralization (K110M, K110F, D197N and D197F), but not charge conserving mutation (K110R and D197E), attenuated the inhibition of currents by protons. CONCLUSIONS AND IMPLICATIONS: Functional inhibition of rat P2X(7) receptors by acidic pH was variably affected by the extra-cellular His(85), Lys(110), Lys(137), Asp(197) and His(219) residues, with the Asp(197) residue being most critical for this inhibition.


Subject(s)
Amino Acids/genetics , Purinergic P2 Receptor Antagonists , Animals , Cell Line , Extracellular Space/metabolism , Humans , Hydrogen-Ion Concentration , Mice , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Point Mutation , Protein Structure, Tertiary , Rats , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2/physiology , Receptors, Purinergic P2X7
5.
Br J Pharmacol ; 151(1): 115-27, 2007 May.
Article in English | MEDLINE | ID: mdl-17351655

ABSTRACT

BACKGROUND AND PURPOSE: The pro-inflammatory cytokine, interleukin-1beta (IL-1beta), has been implicated in the pathogenesis of atherosclerosis, potentially via its release from vascular endothelium. Endothelial cells (EC) synthesize IL-1beta in response to inflammatory stimuli, but the demonstration and mechanism of release of IL-1 from ECs remains unclear. In activated monocytes, efficient release of bioactive IL-1beta occurred via activation of ATP-gated P2X(7) receptors (P2X(7)Rs). Activation of P2X(7)R in ECs from human umbilical vein (HUVECs) released IL-1 receptor antagonist (IL-1Ra). The purpose of this study was to provide a quantitative investigation of P2XR expression and function, in parallel with IL-1beta and IL-1Ra synthesis, processing and release, in HUVECs under pro-inflammatory conditions. EXPERIMENTAL APPROACH: Quantitative RT-PCR, immunoblotting, ELISA, flow cytometry, and whole-cell patch clamp recordings were used to determine protein expression and receptor function. IL-8-luciferase-reporter was used as an IL-1 sensitive bioassay. KEY RESULTS: HUVECs expressed P2X(4)R and P2X(7)R subtypes and both were significantly up-regulated under inflammatory conditions. P2X(7)R currents were increased 3-fold by inflammatory stimuli, whereas no P2X(4)R-mediated currents were detected. Caspase-1, but not IL-1beta, was present intracellularly under basal conditions; inflammatory stimuli activated the synthesis of intracellular pro-IL-1beta and increased caspase-1 levels. Activation of P2X(7)Rs resulted in low-level release of bioactive IL-1beta and simultaneous release of IL-1Ra. The net biological effect of release was anti-inflammatory. CONCLUSIONS AND IMPLICATIONS: Endothelial P2X(7)Rs induced secretion of both pro- and anti-inflammatory IL-1 receptor ligands, the balance of which may provide a means for altering the inflammatory state of the arterial vessel wall.


Subject(s)
Endothelial Cells/metabolism , Interleukin 1 Receptor Antagonist Protein/metabolism , Interleukin-1beta/metabolism , Receptors, Purinergic P2/analysis , Endothelial Cells/chemistry , Humans , Membrane Potentials , RNA, Messenger/analysis , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2/physiology , Receptors, Purinergic P2X4 , Receptors, Purinergic P2X7
6.
Br J Pharmacol ; 149(7): 880-7, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17031385

ABSTRACT

BACKGROUND AND PURPOSE: The ATP-gated P2X(7) receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X(7) receptor-mediated dye uptake in human macrophage THP-1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized. EXPERIMENTAL APPROACH: We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1beta release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP). KEY RESULTS: AZ11645373 up to 10 microM, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X(1), rat P2X(2), human P2X(3), rat P2X(2/3), human P2X(4), or human P2X(5) receptors expressed in HEK cells. AZ11645373 inhibited human P2X(7) receptor responses in HEK cells in a non-surmountable manner with K (B) values ranging from 5 - 20 nM, with mean values not significantly different between assays. K (B) values were not altered by removing extracellular calcium and magnesium. ATP-evoked IL-1beta release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC(50) = 90 nM. AZ11645373 was > 500-fold less effective at inhibiting rat P2X(7) receptor-mediated currents with less than 50% inhibition occurring at 10 microM. CONCLUSIONS AND IMPLICATIONS: AZ11645373 is a highly selective and potent antagonist at human but not rat P2X(7) receptors and will have much practical value in studies of human cells.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Imides/pharmacology , Purinergic P2 Receptor Antagonists , Thiazoles/pharmacology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Aniline Compounds , Animals , Benzoxazoles , Calcium Signaling/drug effects , Cell Line , Dose-Response Relationship, Drug , Fluorescent Dyes , Humans , Interleukin-1beta/metabolism , Ion Channel Gating/drug effects , Lipopolysaccharides/pharmacology , Membrane Potentials/drug effects , Monocytes/drug effects , Monocytes/metabolism , Patch-Clamp Techniques , Quinolinium Compounds , Rats , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X7 , Species Specificity , Thiazoles/chemistry , Transfection , Xanthenes
7.
Br J Pharmacol ; 149(3): 261-8, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16940988

ABSTRACT

BACKGROUND AND PURPOSE: The ATP-gated P2X(7) receptor is an unusual ion channel that couples to multiple downstream signalling cascades. We noted differences in mouse cDNA sequences that may indicate polymorphisms; the aim of this study was to compare function and expression of these mouse P2X(7) receptor mutations. EXPERIMENTAL APPROACH: There are three differences in the sequences of P2X(7) cDNA cloned from mouse NTW8 microglial cells or C57 BL/6 mice: [Phe(11),Ala(221),Met(283)]P2X(7) in the former and [Leu(11),Thr(221),Thr(283)]P2X(7) in the latter. We expressed these receptors and measured membrane currents, ethidium uptake, calcium influx and surface membrane expression. We also carried out these assays on the previously described polymorphism observed between C57 BL/6 and Balb/c mice ([Leu(451)]P2X(7) vs [Pro(451)]P2X(7)). KEY RESULTS: Maximum current densities at [Phe(11),Ala(221),Met(283)]P2X(7) were <12% of those at [Leu(11),Thr(221),Thr(283)]P2X(7) without change in the agonist concentration-response. Replacing methionine with threonine at residue 283 yielded a receptor whose properties were the same as [Leu(11),Thr(221),Thr(283)]P2X(7). Replacing T283 in the rat P2X(7) receptor with methionine yielded currents that were <10% of wildtype and no ethidium uptake was associated with its activation. Maximum current densities and agonist EC(50) values were the same at mouse [Thr(283),Leu(451)]P2X(7) and [Thr(283),Pro(451)]P2X(7) but ethidium uptake and Fluo4 fluorescence were significantly reduced at the [Thr(283),Leu(451)]P2X(7) receptor. There was equivalent surface membrane expression of all P2X(7) receptors. CONCLUSIONS: This study has revealed a residue (Thr(283)) in the ectodomain that is critical for P2X(7) receptor function and suggests that the intracellular residue 451 alters downstream signalling independently of ion channel activity.


Subject(s)
Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2/physiology , Adenosine Triphosphate/pharmacology , Animals , Calcium/metabolism , Cells, Cultured , Humans , Mice , Mice, Inbred C57BL , Receptors, Purinergic P2X7 , Signal Transduction , Structure-Activity Relationship , Threonine
8.
Immunity ; 15(5): 825-35, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11728343

ABSTRACT

The proinflammatory cytokine interleukin-1beta (IL-1beta) is a secreted protein that lacks a signal peptide and does not follow currently known pathways of secretion. Its efficient release from activated immune cells requires a secondary stimulus such as extracellular ATP acting on P2X(7) receptors. We show that human THP-1 monocytes shed microvesicles from their plasma membrane within 2-5 s of activation of P2X(7) receptors. Two minutes after such stimulation, the released microvesicles contained bioactive IL-1beta, which only later appeared in the vesicle-free supernatant. We conclude that microvesicle shedding is a major secretory pathway for rapid IL-1beta release from activated monocytes and may represent a more general mechanism for secretion of similar leaderless secretory proteins.


Subject(s)
Interleukin-1/immunology , Interleukin-1/metabolism , Monocytes/immunology , Cell Line , Humans , Microscopy, Electron, Scanning , Monocytes/ultrastructure , Secretory Vesicles/immunology , Secretory Vesicles/ultrastructure
9.
EMBO J ; 20(22): 6347-58, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11707406

ABSTRACT

P2X receptors are ATP-gated ion channels in the plasma membrane, but activation of the P2X7 receptor also leads to rapid cytoskeletal re-arrangements such as membrane blebbing. We identified 11 proteins in human embryonic kidney cells that interact with the rat P2X7 receptor, by affinity purification followed by mass spectroscopy and immunoblotting [laminin alpha3, integrin beta2, beta-actin, alpha-actinin, supervillin, MAGuK, three heat shock proteins, phosphatidylinositol 4-kinase and receptor protein tyrosine phosphatase-beta (RPTPbeta)]. Activation of the P2X7 receptor resulted in its dephosphorylation. Whole-cell recordings from cells expressing P2X7 receptors showed that this markedly reduced subsequent ionic currents and it also slowed membrane bleb formation. By mutagenesis, we identified Tyr(343) in the putative second transmembrane domain as the site of phosphorylation. Thus, we have identified a P2X7 receptor signalling complex, some members of which may initiate cytoskeletal rearrangements following receptor activation. Others, such as RPTPbeta, might exert feedback control of the channel itself through its dephosphorylation.


Subject(s)
Cell Membrane/metabolism , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/metabolism , Animals , Blotting, Western , Cytoskeleton/metabolism , DNA, Complementary/metabolism , Humans , Immunoblotting , Kidney/embryology , Models, Biological , Mutagenesis, Site-Directed , Mutation , Phosphorylation , Precipitin Tests , Rats , Receptors, Purinergic P2X7 , Signal Transduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time Factors , Transfection , Tyrosine/metabolism
10.
J Acoust Soc Am ; 110(4): 2085-95, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11681386

ABSTRACT

While a large portion of the variance among listeners in speech recognition is associated with the audibility of components of the speech waveform, it is not possible to predict individual differences in the accuracy of speech processing strictly from the audiogram. This has suggested that some of the variance may be associated with individual differences in spectral or temporal resolving power, or acuity. Psychoacoustic measures of spectral-temporal acuity with nonspeech stimuli have been shown, however, to correlate only weakly (or not at all) with speech processing. In a replication and extension of an earlier study [Watson et al., J. Acoust. Soc. Am. Suppl. 1 71. S73 (1982)] 93 normal-hearing college students were tested on speech perception tasks (nonsense syllables, words, and sentences in a noise background) and on six spectral-temporal discrimination tasks using simple and complex nonspeech sounds. Factor analysis showed that the abilities that explain performance on the nonspeech tasks are quite distinct from those that account for performance on the speech tasks. Performance was significantly correlated among speech tasks and among nonspeech tasks. Either, (a) auditory spectral-temporal acuity for nonspeech sounds is orthogonal to speech processing abilities, or (b) the appropriate tasks or types of nonspeech stimuli that challenge the abilities required for speech recognition have yet to be identified.


Subject(s)
Auditory Perception , Individuality , Speech Perception , Adolescent , Adult , Female , Humans , Male , Perceptual Masking , Pitch Discrimination , Psychoacoustics , Reference Values , Speech Acoustics , Speech Discrimination Tests
11.
Percept Psychophys ; 63(4): 737-45, 2001 May.
Article in English | MEDLINE | ID: mdl-11436742

ABSTRACT

The proportion-of-the-total-duration rule (Kidd & Watson, 1992) states that the detectability of a change in a component of a tonal sequence can be predicted by the proportional duration of the changed component relative to the length of the sequence as a whole. A similar viewpoint relies on temporal distinctiveness to account for primacy, recency, and other serial position effects in memory (Murdock, 1960; Neath, 1993a, 1993b). Such distinctiveness models predict that an item will be remembered if it is more distinctive along some dimension relative to possible competitors. Three experiments explored the relation between distinctiveness and proportional duration by examining the effects of the proportion of the total duration of a tone in a sequence, serial position, and interstimulus interval (ISI) on the detection of a change in one component of a tonal sequence. Experiment 1 replicated the basic effect with relatively untrained subjects and a fixed frequency difference. Experiment 2 showed that distinctiveness holds for tonal sequences and a same/different task. Experiment 3 combined the two to show that proportional duration, ISI, and position of the changed tone all contribute to discrimination performance. The present research combines theories that have been proposed in the psychophysics and memory fields and suggests that a comprehensive principle based on relative distinctiveness may be able to account for both perceptual and memory effects.


Subject(s)
Acoustic Stimulation , Auditory Perception , Memory, Short-Term , Serial Learning , Adult , Female , Humans , Male , Models, Psychological , Psychoacoustics
12.
J Biol Chem ; 276(26): 23262-7, 2001 Jun 29.
Article in English | MEDLINE | ID: mdl-11313357

ABSTRACT

ATP-gated P2X(7) purinoceptors are found in most immune cells of the periphery and the brain where their activation leads to multiple downstream events such as cell permeabilization, apoptosis, and/or cytokine release. P2X(7) receptors do not form heteromeric receptors with any of the other six P2X subunits, and it is not known what type of homomeric assemblies the P2X(7) subunit makes. We constructed and purified an ectodomain protein of the rat P2X(7) receptor (amino acids 60-323) and used this to generate a monoclonal antibody (Ab) with which to probe P2X(7) receptors in central and peripheral immune cells. In HEK cells expressing rat P2X(7) receptors, the Ab increased the maximum current evoked by BzATP by 3-8-fold with a 5-fold leftward shift in EC(50) concentration. This Ab recognized only a non-denatured, multimeric form of the receptor on blue native-PAGE but did not recognize the denatured form on SDS-PAGE. A C-terminal polyclonal P2X(7) Ab recognized both monomeric subunits on SDS-PAGE and a multimeric complex on blue native-PAGE in this heterologous expression system. With Western blotting using these two Abs, native P2X(7) receptors in peritoneal macrophage and bone marrow cells are shown to exist as a strongly bound multimeric complex, whereas P2X(7) receptors in brain glia and/or astrocytes appear to form only as monomeric subunits.


Subject(s)
Brain/immunology , Receptors, Purinergic P2/metabolism , Animals , Antibodies, Monoclonal/immunology , Blotting, Western , Bone Marrow Cells/immunology , Cell Extracts/analysis , Electric Conductivity , Electrophoresis, Polyacrylamide Gel , Humans , Immunohistochemistry , Macrophages, Peritoneal/immunology , Protein Denaturation , Protein Subunits , Rats , Rats, Wistar , Receptors, Purinergic P2/immunology , Receptors, Purinergic P2/physiology , Receptors, Purinergic P2X7
13.
J Biol Chem ; 276(18): 14902-8, 2001 May 04.
Article in English | MEDLINE | ID: mdl-11278888

ABSTRACT

The first hydrophobic segment of the rat P2X(2) receptor extends from residue Leu(29) to Val(51). In the rat P2X(2) receptor, we mutated amino acids in this segment and adjoining flanking regions (Asp(15) through Thr(60)) individually to cysteine and expressed the constructs in human embryonic kidney cells. Whole-cell recordings were used to measure membrane currents evoked by brief (2-s) applications of ATP (0.3-100 microM). Currents were normal except for Y16C, R34C, Y43C, Y55C, and Q56C (no currents but normal membrane expression by immunohistochemistry), Q37C (small currents), and F44C (normal current but increased sensitivity to ATP, as well as alphabeta-methylene-ATP). We used methanethiosulfonates of positive, negative, or no charge to test the accessibility of the substituted cysteines. D15C, P19C, V23C, V24C, G30C, Q37C, F44C, and V48C were strongly inhibited by neutral, membrane-permeant methanethiosulfonates. Only V48C was also inhibited by positively and negatively charged methanethiosulfonates, consistent with an extracellular position; however, accessibility of V48C was increased by channel opening. V48C could disulfide with I328C, as shown by the large increase in ATP-evoked current caused by reducing agents. The results suggest that Val(48) at the outer end of the first hydrophobic segment takes part in the gating movement of channel opening.


Subject(s)
Amino Acids/metabolism , Ion Channel Gating , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Line , Disulfides/pharmacology , Humans , Mutagenesis, Site-Directed , Rats , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X2
14.
J Biol Chem ; 275(44): 34190-6, 2000 Nov 03.
Article in English | MEDLINE | ID: mdl-10940304

ABSTRACT

P2X receptor subunits have intracellular N and C termini, two membrane-spanning domains, and an extracellular loop of about 280 amino acids. We expressed the rat P2X(2) receptor in human embryonic kidney cells, and used alanine-scanning mutagenesis on 30 residues with polar side chains conserved among the seven rat P2X receptor subunits. This identified a region proximal to the first transmembrane domain which contained 2 lysine residues that were critical for the action of ATP (Lys(69) and Lys(71)). We substituted cysteines in this region (Asp(57) to Asp(71)) and found that for S65C and I67C ATP-evoked currents were inhibited by methanethiosulfonates. At I67C, the inhibition by negatively charged ethylsulfonate and pentylsulfonate derivatives could be overcome by increasing the ATP concentration, consistent with a reduced affinity of ATP binding. The inhibitory action of the methanethiosulfonates was prevented by pre-exposure to ATP, suggesting occlusion of the binding site. Finally, introduction of negative charges into the receptor by mutagenesis at this position (I67E and I67D) also gave receptors in which the ATP concentration-response curve was right-shifted. The results suggest that residues close to Ile(67) contribute to the ATP-binding site.


Subject(s)
Adenosine Triphosphate/metabolism , Amino Acids/metabolism , Receptors, Purinergic P2/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Immunohistochemistry , Mesylates/antagonists & inhibitors , Mesylates/metabolism , Molecular Sequence Data , Receptors, Purinergic P2/chemistry , Sequence Homology, Amino Acid
15.
Q J Exp Psychol A ; 53(2): 325-48, 2000 May.
Article in English | MEDLINE | ID: mdl-10881609

ABSTRACT

Numerous studies have demonstrated impaired recall when the to-be-remembered information is accompanied or followed by irrelevant information. However, no current theory of immediate memory explains all three common methods of manipulating irrelevant information: requiring concurrent articulation, presenting irrelevant speech, and adding a stimulus suffix. Five experiments combined these manipulations to determine how they interact and which theoretical framework most accurately and completely accounts for the data. In Experiments 1 and 2, a list of auditory items was followed by an irrelevant speech sound (the suffix) while subjects engaged in articulatory suppression. Although articulatory suppression reduced overall recall compared to a control condition, comparable suffix effects were seen in both conditions. Experiments 3 and 4 found reliable suffix effects when list presentation was accompanied by irrelevant speech. Experiment 5 found a suffix effect even when the irrelevant speech was composed of a set of different items. Implications for working memory, precategorical acoustic store, the changing-state hypothesis, and the feature model are discussed.


Subject(s)
Auditory Perception , Cognition , Cues , Mental Recall , Speech , Adult , Female , Humans , Male , Models, Psychological , Perceptual Masking
16.
Annu Rev Pharmacol Toxicol ; 40: 563-80, 2000.
Article in English | MEDLINE | ID: mdl-10836147

ABSTRACT

There are seven P2X receptor cDNAs currently known. Six homomeric (P2X1, P2X2, P2X3, P2X4, P2X5, P2X7) and three heteromeric (P2X2/P2X3, P2X4/P2X6, P2X1/P2X5) P2X receptor channels have been characterized in heterologous expression systems. Homomeric P2X1 and P2X3 receptors are readily distinguishable by their rapid desensitization, the agonist action of alpha beta methyleneATP, and the block by 2',3'-O-(2,4,6-trinitrophenyl)-ATP. P2X2 receptors are unique among homomeric forms in their potentiation by low pH. Homomeric P2X4 receptors are much less sensitive to antagonism by suramin and pyridoxal 5-phosphate-6-azo-2',4'-disulfonic acid. Homomeric P2X7 receptors are the only form in which 2',3'-O-(4-benzoylbenzoyl)-ATP is more potent than ATP. The heteromeric P2X2/P2X3 receptor resembles P2X2 in slow desensitization kinetics and potentiation by low pH and is similar to P2X3 with respect to agonism by alpha beta methyleneATP and block by 2',3'-O-(2,4,6-trinitrophenyl)-ATP. Other agonists, antagonists, and ions that can be used to differentiate among the receptors are discussed.


Subject(s)
Receptors, Purinergic P2/drug effects , Adenosine Triphosphate/pharmacology , Animals , Humans , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2/classification , Recombinant Proteins/drug effects
17.
J Auton Nerv Syst ; 81(1-3): 249-63, 2000 Jul 03.
Article in English | MEDLINE | ID: mdl-10869729

ABSTRACT

P2X receptors are ATP-gated cation channels; they form as homomers or heteromers from a family of seven related subunits. In particular, heteromeric channels comprising P2X(2) and P2X(3) subunits, or P2X(1) and P2X(5) subunits, show distinctive physiological and pharmacological properties in heterologous expression systems. There is substantial evidence that one of the native P2X receptors in sensory neurones corresponds to the P2X(2/3) heteromer, but there is no evidence for P2X(1/5) heteromers in native tissue. We recorded currents in response to activation of heteromeric P2X(1/5) receptors expressed in HEK293 cells to characterize further their functional properties. The ATP concentration-response curve had a threshold concentration of 1 nM, and a Hill slope of one. TNP-ATP was a weak partial agonist, and a non-competitive antagonist which inhibited maximal ATP currents by 60%. Increasing or decreasing pH from 7.3 shifted the ATP concentration-response curves to the right by fivefold and decreased the maximum current by 40%. Calcium permeability was lower than that observed for other P2X receptors (P(Ca)/P(Na) ratio=1.1). The nanomolar sensitivity of this receptor revealed a steady release of ATP from HEK293 cells, providing an extracellular concentration which ranged from 3 to 300 nM. Noradrenaline (0.3-30 microM) increased ATP-evoked currents by 35%; this facilitation occurred within 20 ms. We also recorded excitatory junction potentials (EJPs) from guinea-pig submucosal arterioles. EJPs were inhibited by suramin and PPADS (IC(50)s of 0.2 microM and 20 microM) but TNP-ATP (0.1-10 microM) inhibited EJPs by <30%. Noradrenaline (0.3-30 microM in the presence of phentolamine and propranolol) decreased EJPs in control preparations but facilitated EJPs by 5-20% in submucosal arterioles from reserpinized guinea-pigs. These properties are discussed in relation to P2X receptors underlying EJPs at autonomic neuroeffector junctions.


Subject(s)
Neuromuscular Junction/physiology , Receptors, Purinergic P2/physiology , Animals , Arterioles/anatomy & histology , Arterioles/innervation , Arterioles/physiology , Calcium/pharmacology , Catecholamines/metabolism , Cell Line , Electrophysiology , Guinea Pigs , Histocytochemistry , Hydrogen-Ion Concentration , Male , Membrane Potentials/physiology , Patch-Clamp Techniques , Purinergic P2 Receptor Agonists , Purinergic P2 Receptor Antagonists , Rats , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X , Receptors, Purinergic P2X2 , Receptors, Purinergic P2X5
18.
Mol Pharmacol ; 58(1): 82-8, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10860929

ABSTRACT

There are few antagonists selective for subtypes of the several P2X receptors, but these are needed to identify the receptors expressed on native cells and tissues. In particular, P2X(4) and P2X(7) receptor subunits are colocalized on immune, epithelial, and exocrine gland cells, but both are relatively insensitive to suramin and pyridoxal-5-phosphate-6-azo-2',4'-disulfonic acid derivative. In this article, we show that Coomassie Brilliant Blue G selectively inhibits P2X(7) receptors with nanomolar affinity. We measured currents in response to P2X receptor activation in HEK293 cells heterologously expressing human or rat P2X(1), P2X(2), P2X(3), P2X(2/3), P2X(4), P2X(1/5), and P2X(7) receptors. Brilliant Blue G produced a noncompetitive inhibition of rat and human P2X(7) receptors with IC(50) values of 10 and 200 nM, respectively. IC(50) values for inhibition of the other receptors ranged from 2 to >30 microM; the rat and human P2X(4) receptors showed IC(50) values of >10 and 3.2 microM. Coomassie Blue G also blocked YO-PRO1 uptake and membrane blebbing, which are uniquely associated with activation of P2X(7) receptors. Thus, Brilliant Blue G is at least 1000-fold more potent at rat P2X(7) receptors than at rat P2X(4) receptors.


Subject(s)
Adenosine Triphosphate/metabolism , Purinergic P2 Receptor Antagonists , Rosaniline Dyes/pharmacology , Animals , Benzoxazoles , Biological Transport , Cell Membrane/drug effects , Cell Membrane/physiology , Cells, Cultured , Electrophysiology , Fluorescent Dyes/metabolism , Humans , Ion Channel Gating , Quinolinium Compounds , Rats , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2/physiology , Receptors, Purinergic P2X4 , Receptors, Purinergic P2X7
19.
Mol Pharmacol ; 56(5): 973-81, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10531403

ABSTRACT

P2X receptors are membrane proteins that incorporate a cation-selective ion channel that can be opened by the binding of extracellular ATP. They associate as hetero- and homo-multimers of currently unknown stoichiometry. In this study, we have used Xenopus laevis oocytes to express rat P2X(2) receptor subunits, which carry a cysteine mutation at position 336. ATP-induced currents at this mutant receptor subunit were blocked by more than 90% when exposed to [2-(trimethylammonium) ethyl] methanethiosulfonate (MTSET), whereas currents from wild-type subunits were not affected. To compare mutant and wild-type channel expression, we introduced an epitope in their extracellular domains and found for both channels a similar linear relationship between antibody binding and currents induced by ATP. To study the contribution of the individual subunits to the block by MTSET, we coinjected different mixtures of wild-type and mutant-encoding mRNAs. We found that the inhibition by MTSET depended linearly on the proportion of mutant subunits, which was clearly contrary to the hypothesis that a single mutant subunit could act in a dominant fashion. Subsequent concatenation of wild-type and mutant-encoding cDNAs resulted in an inhibition by MTSET that also depended linearly on the number of mutant subunits and was independent of the position of the mutant subunit, as long as only two or three P2X(2) subunits were joined. With four or six subunits joined, however, the inhibition by MTSET became strongly position-dependent. The present results show that a "per-subunit" channel block causes the blocking effects of MTSET and they suggest that not four but maximally three subunits actively participate in the channel formation.


Subject(s)
Mesylates/pharmacology , Receptors, Purinergic P2/metabolism , Animals , DNA, Complementary/genetics , DNA, Complementary/metabolism , Mutation , Oocytes/drug effects , Oocytes/metabolism , Rats , Receptors, Purinergic P2/biosynthesis , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X2 , Xenopus laevis
20.
J Physiol ; 519 Pt 2: 335-46, 1999 Sep 01.
Article in English | MEDLINE | ID: mdl-10457053

ABSTRACT

1. Extracellular ATP acting on P2X7 receptors opens a channel permeable to small cations, creates an access pathway for the entry of larger molecular weight dyes, and causes cell death. We used whole-cell recording and fluorescence microscopy to measure the time courses of ionic currents, uptake of the propidium dye YO-PRO-1, and membrane disruption, in human embryonic kidney (HEK293) cells expressing the rat P2X7 receptor. 2. The ATP analogue 2', 3'-O-(benzoyl-4-benzoyl)-ATP (30 microM) induced membrane blebbing within 30-40 s of sustained application; this was 5-10 times slower when extracellular sodium was replaced by larger cations. 3. Fluorescence of YO-PRO-1 was detectable within 3 s, and the uptake reached a steady rate within 10-20 s; YO-PRO-1 uptake was greatly enhanced by removing extracellular sodium. 4. Electrophysiological measurements of current reversal potentials with intracellular sodium and extracellular cations of different sizes showed that the ionic channel progressively t'2+LE0i%-i"dilated during 10-20 s to a diameter greater than 1 nm (10 A). With short agonist applications (3-5 s) the pore dilatation and YO-PRO-1 uptake were reversible and repeatable. 5. Polyethylene glycols having molecular weights >= 5000 blocked the increase in cation permeability, YO-PRO-1 uptake and membrane blebbing. 6. We conclude that maximum P2X7 receptor activation causes an exponential dilatation of the ion channel with a time constant of 25 s to a final diameter of 3-5 nm from an initial minimum pore diameter of 0.8 nm.


Subject(s)
Cell Membrane Permeability/physiology , Receptors, Purinergic P2/physiology , Adenosine Triphosphate/antagonists & inhibitors , Adenosine Triphosphate/pharmacology , Animals , Benzoxazoles , Biotransformation/drug effects , Biotransformation/genetics , Biotransformation/physiology , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Membrane Permeability/genetics , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Coloring Agents , Electric Stimulation , Electrophysiology , Fluorescent Dyes , Immunohistochemistry , Kinetics , Microscopy, Fluorescence , Molecular Weight , Patch-Clamp Techniques , Polyethylene Glycols/pharmacology , Quinolinium Compounds , Rats , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X7
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