Joseph Simon

Cloning and functional expression of a brain G-protein-coupled ATP receptor

A cDNA encoding a novel member of the G‐protein‐coupled receptor (GCR) superfamily, an ATP receptor, has been isolated from an embryonic chick whole brain cDNA library by hybridization screening. The encoded protein has a sequence of 362 amino acids (41 kDa) and shares no more than 27% amino acid identity with any known GCR. When expressed as a complementary RNA (cRNA) in Xenopus oocytes a slowly‐developing inward current was observed in response to application of ATP. The pharmacology of this expressed protein defines it as a P2Y purinoceptor.

Cloning and functional characterisation of the mouse P2X7 receptor

We have isolated a 1785‐bp complementary DNA (cDNA) encoding the murine P2X7 receptor subunit from NTW8 mouse microglial cells. The encoded protein has 80% and 85% homology to the human and rat P2X7 subunits, respectively. Functional properties of the heterologously expressed murine P2X7 homomeric receptor broadly resembled those of the P2X7 receptor in the native cell line. However, marked phenotypic differences were observed between the mouse receptor, and the other P2X7 receptor orthologues isolated with respect to agonist and antagonist potencies, and the kinetics of formation of the large aqueous pore.

Characterization and channel coupling of the P2Y(12) nucleotide receptor of brain capillary endothelial cells.

Rat brain capillary endothelial (B10) cells express an unidentified nucleotide receptor linked to adenylyl cyclase inhibition. We show that this receptor in B10 cells is identical in sequence to the P2Y12 ADP receptor (“P2Y T ”) of platelets. When expressed heterologously, 2-methylthio-ADP (2-MeSADP; EC50, 2 nm), ADP, and adenosine 5′-O-(2-thio)diphosphate were agonists of cAMP decrease, and 2-propylthio-d-β,γ-difluoromethylene-ATP was a competitive antagonist (K B , 28 nm), as in platelets. However, 2-methylthio-ATP (2-MeSATP) (EC50, 0.4 nm), ATP (1.9 μm), and 2-chloro-ATP (190 nm), antagonists in the platelet, were also agonists. 2-MeSADP activated (EC50, 0.1 nm) GIRK1/GIRK2 inward rectifier K+ channels when co-expressed with P2Y12 receptors in sympathetic neurons. Surprisingly, P2Y1 receptors expressed likewise gave that response; however, a full inactivation followed, absent with P2Y12 receptors. A new P2Y12-mediated transduction was found, the closing of native N-type Ca2+channels; again both 2-MeSATP and 2-MeSADP are agonists (EC50, 0.04 and 0.1 nm, respectively). That action, like their cAMP response, was pertussis toxin-sensitive. The Ca2+ channel inhibition and K+ channel activation are mediated by βγ subunit release from a heterotrimeric G-protein. Gα subunit types in B10 cells were also identified. The presence in the brain capillary endothelial cell of the P2Y12 receptor is a significant extension of its functional range.

Functional characterization of the P2X4 receptor orthologues

The aim of this study was to functionally characterize the recombinant mouse P2X4 receptor and to compare its pharmacological properties with those of the human and rat orthologues. Whole cell recordings were made from rafts of HEK‐293 cells stably expressing recombinant mouse, rat or human P2X4 receptors, using Cs‐aspartate containing electrodes (3–8 MΩ) in a HEPES‐buffered extracellular medium. The agonist potency of ATP at the three species orthologues was similar, with mean EC50 values of 2.3 μM, 1.4 μM and 5.5 μM, respectively. Adenosine‐5′‐tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X4 receptors (EC50=2.6 and 3.0 μM), but was significantly less potent at the rat orthologue (EC50=20.0 μM). α,β‐methylene adenosine‐5′‐triphosphate (α,β‐meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X4 and 24% at the human P2X4 receptor. In contrast to the other species orthologues, α,β‐meATP failed to elicit a significant agonist response at rat P2X4 receptors, and was found to act as an antagonist, with an IC50 of 4.6 μM, against 10 μM ATP. Mouse P2X4 receptors were found to be sensitive to the antagonist, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (IC50=10.5 μM), as were human P2X4 receptors (IC50=9.6 μM). The rat receptor however, showed a low sensitivity to PPADS (IC50>100 μM). All three orthologues were relatively suramin‐insensitive (IC50>100 μM) and insensitive to 1‐[N,O‐Bis(5‐isoquinoline sulphonyl)benzyl]‐2‐(4‐phenylpiperazine)ethyl]‐5‐isoquinoline sulphonamide (KN‐62; IC50>3 μM). Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X4 receptor, and differ markedly from the rat receptor.

ATP acts via P2Y1 receptors to stimulate acetylcholinesterase and acetylcholine receptor expression: transduction and transcription control.

At the vertebrate neuromuscular junction ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, the P2Y1 receptor, is localized at the junction, and we propose that this mediates a trophic role for synaptic ATP there. Evidence in support of this and on its mechanism is given here. With the use of chick or mouse myotubes expressing promoter–reporter constructs from genes of acetylcholinesterase (AChE) or of the acetylcholine receptor subunits, P2Y1 receptor agonists were shown to stimulate the transcription of each of those genes. The pathway to activation of the AChE gene was shown to involve protein kinase C and intracellular Ca 2+ release. Application of dominant-negative or constitutively active mutants, or inhibitors of specific kinases, showed that it further proceeds via some of the known intermediates of extracellular signal-regulated kinase phosphorylation. In both chick and mouse myotubes this culminates in activation of the transcription factor Elk-1, confirmed by gel mobility shift assays and by the nuclear accumulation of phosphorylated Elk-1. All of the aforementioned activations by agonist were amplified when the content of P2Y1 receptors was boosted by transfection, and the activations were blocked by a P2Y1-selective antagonist. Two Elk-1 binding site sequences present in the AChE gene promoter were jointly sufficient to drive ATP-induced reporter gene transcription. Thus ATP regulates postsynaptic gene expression via a pathway to a selective transcription factor activation.

Functional characterization of the P2X(4) receptor orthologues.

The aim of this study was to functionally characterize the recombinant mouse P2X4 receptor and to compare its pharmacological properties with those of the human and rat orthologues. Whole cell recordings were made from rafts of HEK‐293 cells stably expressing recombinant mouse, rat or human P2X4 receptors, using Cs‐aspartate containing electrodes (3–8 MΩ) in a HEPES‐buffered extracellular medium. The agonist potency of ATP at the three species orthologues was similar, with mean EC50 values of 2.3 μM, 1.4 μM and 5.5 μM, respectively. Adenosine‐5′‐tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X4 receptors (EC50=2.6 and 3.0 μM), but was significantly less potent at the rat orthologue (EC50=20.0 μM). α,β‐methylene adenosine‐5′‐triphosphate (α,β‐meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X4 and 24% at the human P2X4 receptor. In contrast to the other species orthologues, α,β‐meATP failed to elicit a significant agonist response at rat P2X4 receptors, and was found to act as an antagonist, with an IC50 of 4.6 μM, against 10 μM ATP. Mouse P2X4 receptors were found to be sensitive to the antagonist, pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (IC50=10.5 μM), as were human P2X4 receptors (IC50=9.6 μM). The rat receptor however, showed a low sensitivity to PPADS (IC50>100 μM). All three orthologues were relatively suramin‐insensitive (IC50>100 μM) and insensitive to 1‐[N,O‐Bis(5‐isoquinoline sulphonyl)benzyl]‐2‐(4‐phenylpiperazine)ethyl]‐5‐isoquinoline sulphonamide (KN‐62; IC50>3 μM). Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X4 receptor, and differ markedly from the rat receptor.

An elusive receptor is finally caught: P2Y12, an important drug target in platelets

Despite intensive research, the nucleotide P2 receptor that is involved in the aggregation and activation of platelets by ADP has remained elusive. However, now two research groups have independently identified a new platelet receptor of unexpected structure, P2Y(12), that acts with the P2Y(1) receptor to form the site of ADP activation and explains the multiple transduction mechanisms observed in response to ADP in platelets. Recent evidence also suggests that a third component, ATP action on the P2X(1) receptor ion channel, contributes to platelet activation.

Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells

1 CHO‐K1 cells were examined for their cellular responses to the P2 receptor agonist, 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)‐ATP (DbATP), and for the presence of mRNA for P2X receptors. 2 Reverse transcriptase‐polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1‐P2X6 subunits. 3 DbATP (EC50∼100 μm) evoked non‐desensitizing inward currents which reversed at ∼0mV, suggesting activation of a non‐selective cation channel. ATP also evoked inward currents but was less potent than DbATP. 4 DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO‐PRO‐1, in CHO‐K1 cells. Both responses were inhibited by NaCl and MgCl2. In 280 mm sucrose buffer, 45Ca2+ accumulation was measurable within 10–20 s of agonist addition, whereas YO‐PRO‐1 accumulation was only detectable after 8 min. ATP and ATPγS were also agonists but were less potent than DbATP, while UTP, 2‐methylthio ATP, ADP and αβmethylene ATP were inactive at concentrations up to 100 μm. 5 DbATP increased lactate dehydrogenase release from CHO‐K1 cells, suggesting cell lysis, although this effect was only pronounced after 60–90 min. 6 These data suggest that CHO‐K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO‐PRO‐1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.

Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells

Pathological cellular hallmarks of Duchenne muscular dystrophy (DMD) include, among others, abnormal calcium homeostasis. Changes in the expression of specific receptors for extracellular ATP in dystrophic muscle have been recently documented: here, we demonstrate that at the earliest, myoblast stage of developing dystrophic muscle a purinergic dystrophic phenotype arises. In myoblasts of a dystrophin‐negative muscle cell line established from the mdx mouse model of DMD but not in normal myoblasts, exposure to extracellular ATP triggered a strong increase in cytoplasmic Ca2+ concentrations. Influx of extracellular Ca2+ was stimulated by ATP and BzATP and inhibited by zinc, Coomassie Brilliant Blue‐G, and KN‐62, demonstrating activation of P2X7 receptors. Significant expression of P2X4 and P2X7 proteins was immunodetected in dystrophic myoblasts. Therefore, full‐length dystrophin appears, surprisingly, to play an important role in myoblasts in controlling responses to ATP. Our results suggest that altered function of P2X receptors may be an important contributor to pathogenic Ca2+ entry in dystrophic mouse muscle and may have implications for the pathogenesis of muscular dystrophies. Treatments aiming at inhibition of specific ATP receptors could be of a potential therapeutic benefit.‐Yeung, D., Zabłocki, K., Lien, C.‐F., Jiang, T., Arkle, S., Brutkowski, W., Brown, J., Lochmuller, H., Simon, J., Barnard, E. A., Górecki, D. C. Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells. FASEB J. 20, 610–620 (2006)

Coupling of the nucleotide P2Y4 receptor to neuronal ion channels

G protein‐linked P2Y nucleotide receptors are known commonly to stimulate the phosphoinositide signalling pathway. However, we have previously demonstrated that the cloned P2Y2, P2Y6 and P2Y1 receptors couple to neuronal N‐type Ca2+ channels and to M‐type K+ channels. Here we investigate the coupling of recombinant, neuronally expressed rat‐ and human P2Y4 receptors (rP2Y4, hP2Y4) to those channels. Rat sympathetic neurones were nuclear‐injected with a P2Y4 cDNA plasmid. A subsequent activation of rP2Y4 or hP2Y4 by UTP (100 μM) in whole‐cell (ruptured‐patch) mode produced only about 12% inhibition of the N‐type Ca2+ current (ICa(N)). Surprisingly, in perforated patch mode, UTP produced much more inhibition of ICa(N) (maximally 51%), with an IC50 value of 273 nM. This inhibition was voltage‐dependent and was blocked by co‐expression of the βγ‐binding transducin Gα‐subunit. Pertussis toxin (PTX) pretreatment also suppressed ICa(N) inhibition. UTP inhibited the M‐current, recorded in perforated patch mode, by (maximally) 52%, with IC50 values of 21 nM for rP2Y4 and 28 nM for hP2Y4. This inhibition was not affected by PTX pretreatment. With rP2Y4, ATP inhibited the M‐current (IC50 524 nM, 26 times weaker than UTP), whereas ATP had no agonist activity at hP2Y4. This suggests a difference in agonist binding site between rP2Y4 and hP2Y4. We conclude that, in contrast to other nucleotide receptors studied, the P2Y4 receptor couples much more effectively to M‐type K+ channels than to Ca2+ channels. Coupling to the Ca2+ channels involves the βγ‐subunits of Gi/o‐proteins and requires a diffusible intracellular component that is lost in ruptured‐patch recording.