Functional characterization of the P2X<sub>4</sub> receptor orthologues

Jones, Clare A.; Chessell, Iain P.; Simon, József; Barnard, E.A.; Miller, Kerry J.; Michel, Anton D.; Humphrey, P. P. A.

doi:10.1038/sj.bjp.0703059

Cited by 94 publications

(101 citation statements)

References 30 publications

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“…Then, C8-B4 cells were incubated for 30 min at 4°C under gentle rocking with membraneimpermeant EZ-link sulfo-NHS-SS-biotin dissolved at 0.5 mg/ml Toulmé et al, 2006;Casas-Pruneda et al, 2009;Shinozaki et al, 2009). Pharmacological experiments also revealed some distinctive features of P2X4 receptors: ivermectin (IVM) was an allosteric modulator of P2X4 receptors (Khakh et al, 1999b;Priel and Silberberg, 2004), they were relatively resistant to P2X antagonists (Buell et al, 1996;Jones et al, 2000), and antidepressants reduced P2X4 responses (Nagata et al, 2009). Additionally, P2X4 receptors form functional heteromers with P2X6 subunits (Lê et al, 1998a;Ormond et al, 2006) and undergo interactions with P2X7 receptors (Guo et al, 2007;Nicke, 2008;Alqallaf et al, 2009;Boumechache et al, 2009;Casas-Pruneda et al, 2009;MurrellLagnado, 2009).…”

Section: Surface Biotinylation Of P2x4 Receptorsmentioning

confidence: 99%

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

Toulmé¹,

Garcia²,

Samways³

et al. 2010

J Cell Biol

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We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2-6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by 3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking.

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Section: Surface Biotinylation Of P2x4 Receptorsmentioning

confidence: 99%

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

Toulmé¹,

Garcia²,

Samways³

et al. 2010

J Cell Biol

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confidence: 99%

“…Compared with ATP, α,β-methylene ATP (α,β-meATP), in which the oxygen atom linking the α-and β-phosphorous atoms of ATP is replaced by a methylene group (Fig. S1A), reportedly induces a lower maximum current in cells expressing the mouse, rat, and human P2X 4 receptors and other P2X receptors (16,17).The crystal structures of zebrafish P2X 4 receptor (zfP2X 4 ) (18, 19), together with mutational analyses (20-26), provided the structural basis for the channel opening of P2X receptors upon ATP binding. In the crystal structures, zfP2X 4 forms a homotrimer (27,28), in which the transmembrane region of each subunit is composed of two helices (19).…”

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confidence: 99%

Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region

Minato

Suzuki

Hara

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cationselective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X 4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X 4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s −1 ), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.NMR | membrane proteins | ligand-gated ion channels | insect cell expression system | purinergic receptors I n chemical neurotransmission, various neurotransmitters bind to ligand-gated ion channels expressed in the plasma membrane of postsynaptic cells, such as the NMDA, AMPA, and P2X receptors, leading to changes in membrane potential and the concentration of intracellular ions. Each ligand for a ligand-gated ion channel has a distinct ability to evoke currents (1), and the ligands are classified according to the evoked current level: such as, full agonists, partial agonists, and antagonists. Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in antidepressant and smoking-cessation treatment (2, 3).Two mechanisms have been proposed for the partial activation of the ligand-gated ion channels: the equilibrium between the open and closed conformations and the distinct conformation of the partial agonist-bound states from the closed and open conformations (4, 5). In the crystal structures of the extracellular region of the AMPA receptor, in which the distances between the two extracellular domains are changed upon agonist binding, the interdomain distances in the partial agonist-bound states correlated with the conductance level, suggesting that the AMPA receptor adopts specific intermediately permeable conformations (4, 6).The P2X receptors are a family of cation channe...

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“…However, there is a current dearth of such molecules. P2X4 receptors are relatively insensitive to broad-spectrum P2X antagonists, suramin and PPADS [3,14], and although a specific P2X4 antagonist has been reported [39], data regarding its effectiveness and specificity have not yet been published. Tri-cyclic antidepressant drugs such as paroxetine, fluoxetine, desipramine and amitriptyline have been prescribed for many years for the treatment of neuropathic pain [40] and were recently shown to inhibit P2X4 receptor function [15], a mechanism which may contribute to their analgesic properties.…”

Section: Discussionmentioning

confidence: 99%

“…Ivermectin is an allosteric modulator of P2X4 currents, potentiating the peak current and increasing the duration of currents evoked by brief ATP applications [12,13]. There are no specific agonists, and broad-spectrum P2X antagonists are relatively insensitive at P2X4 receptors [3,14]. Recently, antidepressant drugs such as paroxetine, fluoxetine, desipramine and amitriptyline, which are used to treat neuropathic pain, have been proposed to act as P2X4 antagonists [15], though it now appears that they are not true antagonists and have no effect on currents mediated by P2X4 receptors [16,17].…”

Section: Introductionmentioning

confidence: 99%

The carbon monoxide donor, CORM-2, is an antagonist of ATP-gated, human P2X4 receptors

Wilkinson

Kemp

2011

Purinergic Signalling

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Carbon monoxide (CO) is produced endogenously by heme oxygenase (HO) enzymes. HO-1 is highly expressed in many inflammatory disease states, where it is broadly protective. The protective effects of HO-1 expression can be largely mimicked by the exogenous application of CO and CO-releasing molecules (CORMs). Despite a dearth of pharmacological tools for their study, molecular methodologies have identified P2X4 receptors as a potential anti-nociceptive drug target. P2X4 receptors are up-regulated in animal models of inflammatory pain, and their knockdown reduces pain behaviours. In these same animal models, HO-1 expression is anti-nociceptive, and we therefore investigated whether P2X4 was a target for CO and tricarbonyldichlororuthenium (II) dimer (CORM-2). Using conventional whole-cell and perforated-patch recordings of heterologously expressed human P2X4 receptors, we demonstrate that CORM-2, but not CO gas, is an inhibitor of these channels. We also investigated the role of soluble guanylate cyclase and mitochondria-derived reactive oxygen species using pharmacological inhibitors but found that they were largely unable to affect the ability of CORM-2 to inhibit P2X4 currents. A control breakdown product of CORM-2 was also without effect on P2X4. These results suggest that P2X4 receptors are not a molecular target of endogenous CO production and are, therefore, unlikely to be mediating the anti-nociceptive effects of HO-1 expression in inflammatory pain models. However, these results show that CORM-2 is an effective antagonist at human P2X4 receptors and represents a useful pharmacological tool for the study of these receptors given the current dearth of antagonists.

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Functional characterization of the P2X₄ receptor orthologues

Cited by 94 publications

References 30 publications

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region

The carbon monoxide donor, CORM-2, is an antagonist of ATP-gated, human P2X4 receptors

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Functional characterization of the P2X4 receptor orthologues

Cited by 94 publications

References 30 publications

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

Conductance of P2X 4 purinergic receptor is determined by conformational equilibrium in the transmembrane region

The carbon monoxide donor, CORM-2, is an antagonist of ATP-gated, human P2X4 receptors

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Functional characterization of the P2X₄ receptor orthologues

Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region