ATP Concentration-response Curve Research Articles

ATP is not involved in α1-adrenoceptor-mediated vasoconstriction in resistance arteries

Recent publications suggest that α1-adrenoceptor stimulation by exogenous agonists such as phenylephrine in resistance arteries cause contraction through the release of ATP from within the vascular smooth muscle cells. This ATP exits the cell through pannexin-1 channels to act back “autocrine-like” on P2 receptors on the smooth muscle that cause the contraction. In this work we directly test this hypothesis by using a selective P2X1 purinoceptor antagonist NF449 (1–10µM) against phenylephrine and ATP concentration–response curves in small mesenteric arteries of the rat and thoracodorsal arteries of the mouse. We show that NF449 is a simple competitive antagonist of ATP with a pKB of 6.43 and 6.41 in rat and mouse arteries, respectively, but did not antagonise phenylephrine concentration–response curves. This work cautions against the growing overstated role of the reputed pannexin-1/ATP release axis following α1-adrenoceptor activation in small resistance arteries.

Conserved extracellular cysteines differentially regulate the potentiation produced by Zn2+ in rat P2X4 receptors

One feature of the amino acid sequence of P2X receptors identified from mammalian species, Xenopus laevis and zebrafish is the conservation of ten cysteines in the extracellular loop. Little information is available about the role of these conserved ectodomain cysteines in the function of P2X receptors. Here, we investigated the possibility that ten conserved cysteine residues in the extracellular loop of the rat P2X4 receptor may regulate zinc potentiation of the receptor using a series of individual cysteine to alanine point mutations and functional characterization of recombinant receptors expressed in Xenopus oocytes. For the C116A, C132A, C159A, C165A, C217A and C227A mutants, 10µM zinc did not significantly affect the current activated by an EC40 concentration of ATP. By contrast, 5µM zinc shifted the ATP concentration-response curve to the right in a parallel manner for both the C261A and C270A mutants and the magnitudes of those shifts were similar to that of the wildtype receptor. Interestingly, for the C126A and C149A mutants, 5µM zinc potentiated ATP-activated current, but increased the maximal response to ATP by 90% and 81% respectively, without significantly changing the EC50 value of ATP. Thus, these results suggest that cysteines and disulfide bonds between cysteines are differentially involved in the potentiation of the rat P2X4 receptor by zinc.

P2X7 receptors contribute to the currents induced by ATP in guinea pig intestinal myenteric neurons

The whole-cell configuration, several pharmacological tools, and single-cell RT-PCR were used to investigate the contribution of P2X7 subunits to the ATP-induced currents (I ATP) in guinea pig myenteric neurons. I ATP was recorded in the great majority of tested neurons. ATP concentration-response curve (0.01–10 mM) showed two phases, the first mediated by high-sensitive P2X receptors (hsP2X receptors), observed between 0.01–0.3 mM and the second mediated by low-sensitive P2X receptors (lsP2X receptors). The calculated EC 50 values of these phases were 38 and 1759 μM, respectively. 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP) concentration-response curve was monophasic (0.01–1 mM), and less potent (EC 50 142 μM) than ATP to activate hsP2X receptors. A strong inward rectification was noticed when hsP2X receptors were activated with ATP (0.1 mM) and for BzATP-induced currents (0.1 mM; I BzATP) but a significant lower rectification was noticed when lsP2X receptors were activated (5 mM). Brilliant blue G (BBG) at a concentration of 0.3 μM (known to inhibit only P2X7 receptors) reduced I ATP when lsP2X receptors contributed to it but neither affect hsP2X receptors nor I BzATP. However, hsP2X receptors and I BzATP were both inhibited by concentrations ≥ 1 μM of this antagonist. BzATP inhibited hsP2X receptors and therefore, it behaves as partial agonist on these receptors. Using the single-cell RT-PCR technique P2X7 mRNA was detectable in 7 out of 13 myenteric neurons exhibiting P2X2 mRNA. Altogether, our results show that low-sensitive P2X receptors are likely P2X7, whereas, the high-sensitive P2X channels are probably constituted, at least in part, by P2X2 subunits.

C-terminal Calmodulin-binding Motif Differentially Controls Human and Rat P2X7 Receptor Current Facilitation

P2X(7) receptors (P2X(7)R) are ATP-gated calcium-permeable cationic channels structurally unique among the P2X family by their much longer intracellular C-terminal tail. P2X(7)Rs show several unusual biophysical properties, in particular marked facilitation of currents and leftward shift in agonist affinity in response to repeated or prolonged agonist applications. We previously found the facilitation at rat P2X(7)R resulted from a Ca(2+)-calmodulin-dependent process and a distinct calcium-independent process. However, P2X(7)Rs show striking species differences; thus, this study compared the properties of ATP-evoked facilitation of currents in HEK293 cells transiently expressing the human or rat P2X(7)R as well as rat/human, human/rat chimeric, and mutated P2X(7)Rs. Facilitation at the human P2X(7)R was 5-fold slower than at the rat P2X(7)R. Facilitation did not resulting from an increase of receptor addressing the plasma membrane. We found the human P2X(7)R shows only calcium-independent facilitation with no evidence for calmodulin-dependent processes, nor does it contain the novel 1-5-16 calmodulin binding domain present in the C terminus of rat P2X(7)R. Replacement of three critical residues of this binding domain from the rat into the human P2X(7)R (T541I, C552S, and G559V) reconstituted the Ca(2+)-calmodulin-dependent facilitation, leaving the calcium-independent facilitation unaltered. The leftward shift in the ATP concentration-response curve with repeated agonist applications appears to be a property of the calcium-independent facilitation process because it was not altered in any of the chimeric or mutated P2X(7)Rs. The absence of Ca(2+)-dependent facilitation at the human P2X(7)R may represent a protective adaptation of the innate immune response in which P2X(7)R plays significant roles.

A point mutation in the ectodomain‐transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors

ATP-gated P2X4 receptors (P2X4R) are abundantly expressed in the CNS. However, little is known about the molecular targets for ethanol action in P2X4Rs. The current investigation tested the hypothesis that the ectodomain-transmembrane (TM) interface contains residues that are important for the action of ethanol in P2X4Rs. Wild type (WT) and mutant P2X4R were expressed in Xenopus oocytes. ATP concentration-response curves and ethanol (10-200 mM)-induced changes in ATP EC(10)-gated currents were determined using two-electrode voltage clamp (-70 mV). Alanine substitution at the ectodomain-TM1 interface (positions 50-61) resulted in minimal changes in ethanol response. On the other hand, alanine substitution at the ectodomain-TM2 interface (positions 321-337) identified two key residues (D331 and M336) that significantly reduced ethanol inhibition of ATP-gated currents without causing marked changes in ATP I(max), EC(50), or Hill's slope. Other amino acid substitutions at positions 331 and 336 significantly altered or eliminated the modulatory effects of ethanol. Linear regression analyses revealed a significant relationship between hydropathy and polarity, but not molecular volume/molecular weight of the residues at these two positions. The results support the proposed hypothesis and represent an important step toward developing ethanol-insensitive receptors for investigating the role of P2X4Rs in mediating behavioral effects of ethanol.

Extracellular histidine residues identify common structural determinants in the copper/zinc P2X2 receptor modulation

To assess the mechanism of P2X2 receptor modulation by transition metals, the cDNA for the wild-type receptor was injected to Xenopus laevis oocytes and examined 48-72 h later by the two-electrode voltage-clamp technique. Copper was the most potent of the trace metals examined; at 10 microm it evoked a 25-fold potentiation of the 10 microm ATP-gated currents. Zinc, nickel or mercury required 10-fold larger concentrations to cause comparable potentiations, while palladium, cobalt or cadmium averaged only 12- and 3-fold potentiations, respectively. Platinum was inactive. The non-additive effect of copper and zinc at 10-100 microm suggests a common site of action; these metals also shifted to the left the ATP concentration-response curves. To define residues necessary for trace metal modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X2 receptor. The H120A and H213A mutants were resistant to the modulator action of copper, zinc and other metals with the exception of mercury. Mutant H192A showed a reduction but not an abrogation of the copper or zinc potentiation. H245A showed less affinity for copper while this mutant flattened the zinc-induced potentiation. Mutant H319A reduced the copper but not the zinc-induced potentiation. In contrast, mutants H125A, H146A, H152A and H174A conserved the wild-type receptor sensitivity to trace metal modulation. We propose that His120, His192, His213 and His245 form part of a common allosteric metal-binding site of the P2X2 receptor, which for the specific coordination of copper, but not zinc, additionally involves His319.

The mechanism by which ethanol inhibits rat P2X4 receptors is altered by mutation of histidine 241

1. We investigated ethanol inhibition of the rat P2X(4) receptor and the contribution of the three histidine residues in the extracellular loop of this receptor to ethanol inhibition of receptor function, using site-directed mutagenesis and electrophysiological characterization of recombinant receptors. 2. In the wild-type receptor, 50, 200 and 500 mM ethanol increasingly shifted the ATP concentration-response curve to the right in a parallel manner, increasing the EC(50) value without affecting E(max). However, 750 or 900 mM ethanol did not produce a further increase in the EC(50) value of the ATP concentration-response curve, suggesting that this inhibition is not competitive. 3. The P2X(4) receptor mutations H140A and H286A did not significantly alter ethanol inhibition of ATP-activated current. By contrast, the mutation H241A changed the mechanism by which ethanol inhibits receptor function; viz., ethanol inhibition was not associated with an increased EC(50) value of the ATP concentration-response curve, instead, ethanol decreased the maximal response to ATP without affecting the EC(50) value of the ATP concentration-response curve. 4. Ethanol inhibition of the H241A mutant was voltage independent between -60 and +20 mV and ethanol did not alter the reversal potential of ATP-activated current. In addition, ethanol decreased the desensitization rate of the H241A-mediated current. 5. The purinoceptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), did not alter the magnitude of ethanol inhibition of ATP-activated current in the H241A mutant. 6. The results suggest that ethanol inhibits the wild-type rat P2X(4) receptor by an allosteric action to increase the EC(50) value of the ATP concentration-response curve, the P2X(4) receptor mutation H241A alters the mechanism by which ethanol inhibits P2X(4) receptor function, and ethanol and PPADS or suramin appear to inhibit H241A-mutated receptors at independent sites.

Metabolic Regulation of the Pancreatic Beta-Cell ATP-Sensitive K+ Channel

Closure of ATP-sensitive K+ channels (KATP channels) is a key step in glucose-stimulated insulin secretion. The precise mechanism(s) by which glucose metabolism regulates KATP channel activity, however, remains controversial. It is widely believed that the principal determinants are the intracellular concentrations of the metabolic ligands, ATP and ADP, which have opposing actions on KATP channels, with ATP closing and MgADP opening the channel. However, the sensitivity of the channel to these nucleotides in the intact cell, and their relative contribution to the regulation of channel activity, remains unclear. The precise role of phosphoinositides and long-chain acyl-CoA esters, which are capable of modulating the channel ATP sensitivity, is also uncertain. Furthermore, it is still a matter of debate whether it is changes in the concentration of ATP, of MgADP, or of other agents, which couples glucose metabolism to KATP channel activity. In this article, we review current knowledge of the metabolic regulation of the KATP channel and provide evidence that MgADP (or MgATP hydrolysis), acting at the regulatory subunit of the channel, shifts the ATP concentration-response curve into a range in which the channel pore can respond to dynamic changes in cytosolic ATP. This metabolic pas de deux orchestrates the pivotal role of ATP in metabolic regulation of the KATP channel.

Heavy metals modulate the activity of the purinergic P2X 4 receptor

To further characterize the nature of the regulatory metal-binding sites of the rat P2X 4 receptor, several transition heavy metals were tested to examine their ability to mimic the facilitator action of zinc or the inhibitory action of copper. cDNA coding for the rat P2X 4 receptor was injected into Xenopus laevis oocytes; the two-electrode voltage-clamp technique was used to measure and quantify the ATP-evoked currents in the absence or presence of the metals. Cadmium facilitated the ATP-gated currents in a reversible and voltage-independent manner; maximal potentiation occurred within less than 1 min. Cadmium displaced leftward, in a concentration-dependent manner, the ATP concentration–response curve. In contrast, mercury reduced the ATP-gated currents in a reversible, time, and concentration manner. Maximal inhibition occurred after about 5 min of metal application. Cobalt also augmented the ATP-evoked currents, but its action was long lasting and did not reverse even after 45 min of metal washout. Other metals such as lead, nickel, manganese, silver, or gallium did not significantly alter the ATP-gated currents. The co-application of cadmium plus zinc or mercury plus copper caused additive effects. Mutation of H140 by alanine (H140A) augmented both the cadmium-induced facilitation and the mercury-induced inhibition. In contrast, the H241A mutant showed characteristics indistinguishable from the wild type. The H286A mutant showed a normal cadmium-induced potentiation, but an increased mercury inhibition. Out of the metals examined, only cadmium mimicked closely the action of zinc, evidencing commonalities. While mercury mimicked the action of copper, both metals apparently interact at distinct metal-binding sites. The present findings allow us to infer that heavy metals modulate the P2X 4 receptor by acting in at least three separate metal-binding sites.

ATP-induced endothelium-independent enhancement of lymphatic vasomotion in guinea-pig mesentery involves P2X and P2Y receptors.

1. The present study has investigated mechanisms underlying ATP-induced endothelium-independent enhancement of vasomotion in guinea-pig mesenteric lymphatic vessels. 2. Lymphatic vasomotion, vessel tone and smooth muscle [Ca(2+)](i) showed similar ATP concentration-response curves. 3. ATP, at 0.1 mM, caused a biphasic increase in tonic [Ca(2+)](i) and superimposed vasomotion-associated Ca(2+) transients. All ATP-induced [Ca(2+)](i) changes were abolished by incubating the smooth muscle with suramin (0.1 mM). 4. alpha,beta-MeATP (0.1 mM) and UTP (0.1 mM) caused similar changes in [Ca(2+)](i) but the responses to these agonists were smaller than to ATP. 5. The actions of alpha,beta-MeATP (0.1 mM) were inhibited by suramin (0.1 mM) and PPADS (30 micro M) but not by reactive blue 2 (30 micro M). 6. In the presence of alpha,beta-MeATP (0.1 mM), the increases in tonic [Ca(2+)](i) and vasomotion-associated Ca(2+) transients induced by ATP (0.1 mM) were inhibited by U73122 (5 micro M), CPA (20 micro M) and heparin, whereas U73343 (5 micro M) and pre-treatment with PTx (100 ng ml(-1)) had no significant effects. 7. Depletion of the intracellular stores with CPA (20 micro M) caused an increase in [Ca(2+)](i), which was not blocked by desensitization of P(2X) receptors with alpha,beta-MeATP. 8. The data indicate that ATP, at relatively high concentrations increases lymphatic smooth muscle [Ca(2+)](i) and vasomotion through activation of P(2X1) and P(2Y2) purinoceptors present on lymphatic smooth muscle. The increase in [Ca(2+)](i) is likely to result from Ca(2+) release from inositol-1,4,5-trisphosphate-sensitive stores as well as Ca(2+) influx through store-operated channels and P(2X)-gated channels.

Modulation by oxytocin of ATP-activated currents in rat dorsal root ganglion neurons

The modulatory effect of oxytocin (OT) on ATP-activated currents (I ATP) was studied in freshly isolated dorsal root ganglion (DRG) neurons of rats using whole cell clamp technique. In most of the neurons examined (50/70, 71.4%) extracellular application of OT (10 −9–10 −5 mol/L) suppressed I ATP while in the rest (20/70, 28.6%) no modulatory effect was observed. OT shifted the ATP concentration-response curve downwards with a decrease of 39.8±4.2% in the maximal current response and with no significant change of Kd value. This OT-induced inhibition of I ATP showed no voltage dependence, and could be blocked by [d(CH 2) 5,Tyr(Me) 2,Thr 4,Tyr-NH 2 9]-OVT (d(CH 2) 5-OVT) (10 −8 mol/L), a specific OT receptor antagonist. Intracellular application of H-9 (4×10 −5 mol/L, an inhibitor of protein kinase A) ( n=12), BAPTA (10 −2 mol/L, a chelator of calcium ions) ( n=4) could reverse the inhibitory effect of extracellular OT (10 −7 mol), while inclusion of H-7 (2×10 −5 mol/L, a protein kinase C inhibitior) ( n=8) and KN-93 (10 −5 mol/L, an inhibitor of CaMKII) ( n=9) in the recording pipette did not affect this effect. The results suggested that OT inhibition on ATP-activated currents was mediated by OT receptors in the membrane of DRG neurons; and this inhibitory effect involved the transduction of intracellular cAMP-PKA and Ca 2+.

Polar Residues of the Second Transmembrane Domain Influence Cation Permeability of the ATP-gated P2X2 Receptor

P2X receptors are simple polypeptide channels that mediate fast purinergic depolarizations in both nerve and muscle. Although the depolarization results mainly from the influx of Na(+), these channels also conduct a significant Ca(2+) current that is large enough to evoke transmitter release from presynaptic neurons. We sought to determine the molecular basis of this Ca(2+) conductance by a mutational analysis of recombinant P2X(2) receptors. Wild type and 31 mutant P2X(2) receptors were expressed in HEK-293 cells and studied under voltage-clamp. We found that the relative Ca(2+) permeability measured from the reversal potentials of ATP-gated currents was unaffected by neutralizing fixed charge (Asp(315), Asp(349)) near the mouths of the channel pore. By contrast, mutations that changed the character or side chain volume of three polar residues (Thr(336), Thr(339), Ser(340)) within the pore led to significant changes in P(Ca)/P(Cs). The largest changes occurred when Thr(339) and Ser(340) were replaced with tyrosine; these mutations almost completely abolished Ca(2+) permeability, reduced P(Li)/P(Cs) by about one-half, and shifted the relative permeability sequence of Cs(+), Rb(+), K(+), and Na(+) to their relative mobility in water. Our results suggest that the permeability sequence of the P2X(2) receptor arises in part from interactions of permeating cations with the polar side chains of three amino acids located in a short stretch of the second transmembrane domain.

Differential response of K(ATP) channels containing SUR2A or SUR2B subunits to nucleotides and pinacidil.

ATP-sensitive K-channels (K(ATP) channels) are the target for K(ATP)-channel openers (KCOs), such as pinacidil and P1075. These channels are formed from pore-forming Kir6.2 and regulatory sulfonylurea receptors (SUR2A in heart and skeletal muscle; SUR2B in smooth muscle). The two isoforms of SUR2 differ only in their final 42 amino acids, a region that includes neither the Walker A and B nucleotide binding motifs nor the proposed KCO binding site, yet channels containing SUR2A or SUR2B respond differently to both nucleotides and KCOs. We explored the basis for this difference by expressing Kir6.2/SUR2A and Kir6.2/SUR2B currents in Xenopus laevis oocytes. Kir6.2/SUR2B but not Kir6.2/SUR2A currents were activated by the Mg-nucleoside triphosphates MgATP and MgGTP, whereas both channel types responded to the diphosphates MgADP and MgGDP. This activation of Kir6.2/SUR2B currents by MgATP explains how the ATP concentration-response curve is shifted to the right in the presence of Mg(2+). In the absence of nucleotide, pinacidil and P1075 activated Kir6.2/SUR2B and Kir6.2/SUR2A currents, but the presence of nucleotide slowed the drug off-rates. In the presence of MgATP, the response to pinacidil reversed approximately 14 times more slowly with SUR2B than SUR2A. The EC(50) for ATP, measured by its ability to slow the pinacidil off-rate, was also approximately 20 times higher for channels containing SUR2A than SUR2B. Our findings suggest that nucleotide binding and/or hydrolysis is enhanced in SUR2B compared with SUR2A, and that the greater KCO-affinities of SUR2B compared with SUR2A may be a consequence of this altered nucleotide handling.

Identification of Amino Acid Residues Contributing to the ATP-binding Site of a Purinergic P2X Receptor

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.

Inhibition by ethanol of rat P2X(4) receptors expressed in Xenopus oocytes.

1. The effect of ethanol on the function of P2X(4) receptors expressed in Xenopus oocytes was studied using two-electrode voltage-clamp recording. 2. The amplitude of current activated by 1 microM ATP was decreased by ethanol in a concentration-dependent manner over the concentration range 1 - 500 mM. The concentration of ethanol that produced 50% inhibition (IC(50)) of current activated by 1 microM ATP was 58 mM. 3. Ethanol inhibition of ATP-activated current was not dependent on membrane potential from -60 to +20 mV, and ethanol did not change the reversal potential of ATP-activated current. 4. Ethanol, 50 mM, shifted the ATP concentration-response curve to the right, increasing the EC(50) for ATP from 9.1 to 16.0 microM, but did not reduce the maximal response to ATP. 5. The results suggest that ethanol may inhibit P2X(4) receptors by decreasing the apparent affinity of the binding site for ATP. 6. Since the P2X(4) receptor is the most abundant P2X subunit in the brain, these receptors could be important effectors of ethanol action in the central nervous system.

Zinc and copper modulate differentially the P2X4 receptor.

The rat ATP P2X4 receptor was expressed in Xenopus laevis oocytes to assess the effect of zinc and copper as possible regulators of purinergic mechanisms. ATP applied for 20 s evoked an inward cationic current with a median effective concentration (EC50) of 21.4+/-2.8 microM and a Hill coefficient (nH) of 1.5+/-0.1. Coapplication of ATP plus 10 microM zinc displaced leftward, in a parallel fashion, the ATP concentration-response curve, reducing the EC50 to 8.4+/-1.8 microM (p < 0.01) without altering the receptor nH. The zinc potentiation was fast in onset, easily reversible, and voltage-independent and did not require metal preexposure. The zinc EC50 was 2-5 microM, with a bell-shaped curve. At concentrations of 100-300 microM, zinc produced less potentiation, and at 1 mM, it inhibited 50% the ATP current. The effect of zinc was mimicked by cadmium. In contrast, copper inhibited the ATP-evoked currents in a time- and concentration-dependent fashion, reducing the maximal current (Imax) without altering the EC50. The copper-induced inhibition was slow in onset, slowly reversible, and voltage-independent. Whereas coapplication of 300 microM copper plus ATP reduced Imax to 36.2+/-5%, the coapplication of, or 60-s preexposure by, 10 microM copper reduced Imax to 79+/-9.2% (p < 0.05) and 39.6+/-8.7% (p < 0.01), respectively. The inhibition was noncompetitive in nature and mimicked by mercury. Cobalt, barium, and manganese did not modify significantly the ATP-evoked current, demonstrating metal specificity. The simultaneous 1-min preapplication of both metals revealed that the 10 microM zinc-induced potentiation was obliterated by 10 microM copper, whereas 30 microM copper not only reduced the potentiation, but inhibited the ATP response. Following coapplication of both metals for 20 s with ATP, at least 100 microM copper was required to counteract the 10 microM zinc-induced potentiation. The simultaneous preincubation with both metals provided evidence for a noncompetitive interaction. We hypothesize the existence of metal binding site(s), which are most likely localized in the extracellular domain of the P2X4 receptor structure. These sites are selective and accessible to extracellular metal applications and bind micromolar concentrations of metals. The present results are compatible with the working hypothesis that trace metals, such as copper and zinc, are physiological modulators of the P2X4 receptor. The modulation of brain purinergic transmission by physiologically and toxicologically relevant trace metal cations is highlighted.

Diinosine pentaphosphate (IP5I) is a potent antagonist at recombinant rat P2X1 receptors.

1. The antagonist activity of a series of diinosine polyphosphates (IpnI, where n=3, 4, 5) was assessed against ATP-activated inward currents at rat P2X(1-4) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2. Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap5A into Ip5I) using 5'-adenylic deaminase, and purified using reverse-phase chromatography. 3. Against ATP-responses at rP2X1 receptors, the potency order for antagonism was (pIC50): Ip5I (8.5)>Ip4I (6.3)>Ip3I (>4.5). Ip5I (10-100 nM) caused a concentration-dependent rightwards displacement of the ATP concentration-response curve without reducing the maximum ATP effect. However, the Schild plot was non-linear which indicated Ip5I is not a competitive antagonist. Blockade by micromolar concentrations of Ip5I was not surmountable. Ip4I also behaved as a non-surmountable antagonist. 4. Against ATP-responses at rP2X3 receptors, the potency order for antagonism was (pIC50): Ip4I (6. 0)>Ip5I (5.6)>Ip3I (>4.5). Blockade by Ip4I (pA2, 6.75) and Ip5I (pA2, 6.27) was surmountable at micromolar concentrations. 5. Diinosine polyphosphates failed to inhibit ATP-responses at rP2X2 receptors, whereas agonist responses at rP2X4 were reversibly potentiated by Ip4I and Ip5I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X1 - 4 receptors. 6. Thus, Ip5I acted as a potent and relatively-selective antagonist at the rP2X1 receptor. This dinucleotide pentaphosphate represents a high-affinity antagonist for the P2X1 receptor, at which it acts in a competitive manner at low (</=100 nM) concentrations but has more complex actions at higher (>100 nM) concentrations.

Differential modulation by copper and zinc of P2X2 and P2X4 receptor function.

Differential Modulation by Copper and Zinc of P2X2 and P2X4 Receptor Function. The modulation by Cu2+ and Zn2+ of P2X2 and P2X4 receptors expressed in Xenopus oocytes was studied with the two-electrode, voltage-clamp technique. In oocytes expressing P2X2 receptors, both Cu2+ and Zn2+, in the concentration range 1-130 microM, reversibly potentiated current activated by submaximal concentrations of ATP. The Cu2+ and Zn2+ concentrations that produced 50% of maximal potentiation (EC50) of current activated by 50 microM ATP were 16.3 +/- 0.9 (SE) microM and 19.6 +/- 1.5 microM, respectively. Cu2+ and Zn2+ potentiation of ATP-activated current was independent of membrane potential between -80 and +20 mV and did not involve a shift in the reversal potential of the current. Like Zn2+, Cu2+ increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally effective concentration of Zn2+, suggesting a common site or mechanism of action of Cu2+ and Zn2+ on P2X2 receptors. For the P2X4 receptor, Zn2+, from 0.5 to 20 microM enhanced current activated by 5 microM ATP with an EC50 value of 2.4 +/- 0.2 microM. Zn2+ shifted the ATP concentration-response curve to the left in a parallel manner, and potentiation by Zn2+ was voltage independent. By contrast, Cu2+ in a similar concentration range did not affect ATP-activated current in oocytes expressing P2X4 receptors, and Cu2+ did not alter the potentiation of ATP-activated current produced by Zn2+. The results suggest that Cu2+ and Zn2+ differentially modulate the function of P2X2 and P2X4 receptors, perhaps because of differences in a shared site of action on both subunits or the absence of a site for Cu2+ action on the P2X4 receptor.

Inhibition of ATP‐activated current by zinc in dorsal root ganglion neurones of bullfrog

1. The effect of Zn2+ on ATP-activated current was studied in bullfrog dorsal root ganglion (DRG) neurones using the whole-cell patch-clamp technique. 2. Zn2+ (2-800 microM) inhibited current activated by submaximal concentrations of ATP. The Zn2+ concentration that produced 50% inhibition (IC50) of current activated by 2.5 microM ATP was 61 +/- 9.8 microM. When ATP concentrations were adjusted to account for chelation of Zn2+, the IC50 of Zn2+ was 86 +/- 18 microM. 3. The inhibitory action of Zn2+ on ATP-gated channels did not appear to be due to a decrease in the concentration of one or more species of ATP. 4. Zn2+ inhibition of ATP-activated current was independent of membrane potential between -80 and +40 mV, and did not involve a shift in the reversal potential of the current. 5. Zn2+ (100 microM) shifted the ATP concentration-response curve to the right in a parallel manner, increasing the EC50 for ATP from 2.5 +/- 0.5 microM to 5.5 +/- 0.4 microM. 6. Zn2+ decreased the time constant of deactivation of ATP-gated ion channels without affecting the time constant of activation or desensitization. 7. Dithiothreitol (DTT) reversed Zn2+ inhibition of ATP-activated current. 8. 2-Methylthio ATP, alpha,beta-methylene ATP and ADP activated current with EC50 values of 2.4 +/- 0.3. 50.1 +/- 5.8 and 303.1 +/- 53.9 microM, respectively. Adenosine, AMP or beta,gamma-methylene ATP did not evoke detectable current. 9. Reactive Blue 2 and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid inhibited ATP-activated current. 10. The results suggest that Zn2+ can inhibit P2X purinoceptor function by decreasing the affinity of the binding site for ATP. These observations provide the first evidence for this action of Zn2+ on a neurotransmitter-gated ion channel. Furthermore, the receptor-channel in these neurones appears to be a novel member of the P2X purinoceptor class.

Different sensitivities to pH of ATP-induced currents at four cloned P2X receptors.

The effect of changing extracellular pH was studied on the currents induced by ATP or alphabeta-methylene-ATP in HEK293 cells transfected with different P2X receptor subunits. In cells expressing P2X1, P2X3, or P2X4 receptors, the effect of ATP was decreased by acidification. In cells expressing P2X2 receptors, acidification increased the ATP-induced current; this effect was also seen in cells expressing heteromeric P2X2 and P2X3 receptors. At P2X2 receptors, acidification caused a leftward shift in the ATP concentration-response curve, without change in maximum; the pKa for this effect was 7.3. At P2X4 receptors, acidification caused a rightward shift in the ATP concentration-response curve, without change in the maximum; the pKa for this effect was 6.8. The pH dependence of the action of ATP should be taken into account in studies of synaptic transmission, and it may provide a further tool to assign molecular identity to P2X receptors expressed by brain neurons.