Functional and biochemical evidence for heteromeric ATP-gated channels composed of P2X1 and P2X5 subunits.
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The mammalian P2X receptor gene family encodes two-transmembrane domain nonselective cation channels gated by extracellular ATP. Anatomical localization data obtained by in situ hybridization and immunocytochemistry have shown that neuronal P2X subunits are expressed in specific but overlapping distribution patterns. Therefore, the native ionotropic ATP receptors diversity most likely arises from interactions between different P2X subunits that generate hetero-multimers phenotypically distinct from homomeric channels. Rat P2X1 and P2X5 mRNAs are localized within common subsets of peripheral and central sensory neurons as well as spinal motoneurons. The present study demonstrates a functional association between P2X1 and P2X5 subunits giving rise to hybrid ATP-gated channels endowed with the pharmacology of P2X1 and the kinetics of P2X5. When expressed in Xenopus oocytes, hetero-oligomeric P2X1+5 ATP receptors were characterized by slowly desensitizing currents highly sensitive to the agonist alpha,beta-methylene ATP (EC50 = 1.1 microM) and to the antagonist trinitrophenyl ATP (IC50 = 64 nM), observed with neither P2X1 nor P2X5 alone. Direct physical evidence for P2X1+5 co-assembly was provided by reciprocal subunit-specific co-purifications between epitope-tagged P2X1 and P2X5 subunits transfected in HEK-293A cells. (+info)
Properties of the novel ATP-gated ionotropic receptor composed of the P2X(1) and P2X(5) isoforms.
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We recently reported that a novel hetero-oligomeric P2X receptor is formed from the P2X(1) and P2X(5) isoforms when coexpressed in human embryonic kidney 293 cells (). A more complete description of the pharmacology of this novel receptor is presented here. A brief application of ATP to a voltage-clamped cell transiently expressing P2X(1/5) receptors resulted in a biphasic current that rapidly reached a peak and then decayed to a sustained plateau. Washout of ATP was accompanied by generation and fade of a pronounced tail of inward current. EC(50) values were determined from concentration-response curves for a range of agonists. The rank order of agonist potency was ATP >/= 2 methylthio ATP > adenosine 5'-O-(3-thiotriphosphate) > alpha,beta-methylene ATP > ADP > CTP. alpha,beta-methylene ADP, UTP, GTP, and AMP were ineffective. Only ATP and 2 methylthio ATP were full agonists. IC(50) values were determined from concentration-response curves for three commonly used purinergic antagonists. Suramin and pyridoxal phosphate-6-azophenyl-2', 4'-disulfonic acid were equipotent at P2X(1) and P2X(1/5) receptors; however, the P2X(1/5) receptor was much less sensitive to TNP-ATP than was the P2X(1) receptor. The amplitude of peak ATP-gated current was relatively insensitive to changes in [Ca(2+)](O) (1-30 mM). Finally, plateau currents were potentiated by low concentrations of pyridoxal phosphate-6-azophenyl-2', 4'-disulfonic acid and by raising [Ca(2+)](O). These results provide additional information on the pharmacological profile of the recombinant P2X(1/5) receptor channel and provide a basis to further evaluate ATP-induced currents in native tissues. (+info)
Modulatory activity of extracellular H+ and Zn2+ on ATP-responses at rP2X1 and rP2X3 receptors.
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1 The modulatory activity of extracellular H+ and Zn2+ was examined on ATP-responses at rat P2X1 (rP2X1) and rat P2X3 (rP2X3) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2 Superfused ATP (0.03-30 microM, at pH 7.5) evoked inward currents at rP2X1 receptors (EC50 value, 300+/-7 nM). ATP potency was reduced 2 fold at pH 6.5, and 6 fold at pH 5.5, without altering the maximum ATP effect. Alkaline conditions (pH 8.0) did not alter ATP activity. 3 Superfused ATP (0.01 - 300 microM, at pH 7. 5) evoked inward currents at rP2X3 receptors (EC50 value, 1.8+/-0.3 microM). ATP activity was affected only at pH 5.5, reducing agonist potency 15 fold without altering the maximum ATP effect. 4 Extracellular Zn2+ inhibited ATP-responses at rP2X1 receptors in a time-dependent manner, a 20 min pre-incubation being optimal (IC50 value, 1.0+/-0.2 microM). However, the Zn2+ effect was pH-independent, suggesting Zn2+- and H+-inhibition of ATP-responses occur through independent processes. 5 Extracellular Zn2+ weakly potentiated ATP-responses at rP2X3 receptors (EC50 value, 11+/-1 microM). The Zn2+ effect was dependent on pre-incubation time and, with 20 min pre-incubation periods, Zn2+ potentiated then inhibited ATP-responses in a concentration-dependent, but pH-independent, manner. 6 In summary, ATP activity at rP2X1 receptors was decreased by both extracellular H+ and Zn2+ and their effects were additive. ATP activity at rP2X3 receptors was less sensitive to H+-inhibition and, in contrast, was potentiated by Zn2+ in a pH-independent manner. These differential effects may help distinguish P2X1 and P2X3 receptors in whole tissues. (+info)
Congestive heart failure induces downregulation of P2X1-receptors in resistance arteries.
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OBJECTIVE: Congestive heart failure (CHF) is accompanied by enhanced peripheral sympathetic nerve activity, increased vascular resistance and impaired peripheral blood flow. Besides noradrenaline and neuropeptide Y, the sympathetic nervous system also releases ATP, which has contractile effects mediated by different subtypes of P2-receptors on the vascular smooth muscle cells. The present study was designed to examine postsynaptic changes of the contractile responses to ATP and other extracellular nucleotides in CHF. METHODS: CHF was induced by left coronary artery ligation resulting in a reproducible myocardial infarction in Sprague-Dawley rats. Contractile responses were examined in cylindrical segments of aorta and the mesenteric artery after endothelium removal. To determine if an altered response was regulated on the transcriptional level, competitive reverse transcription polymerase chain reaction (RT-PCR) was used to estimate the amount of P2X1-receptor mRNA. RESULTS: ATP, which is both a P2X1- and a P2Y-receptor agonist, induced a weaker contraction in the mesenteric artery from CHF as compared to sham operated rats. A decrease in both potency and maximum contraction was shown for the selective P2X1-receptor agonist, alpha beta-MeATP, in the mesenteric artery (pEC50 = 6.04 vs. 5.76, Cmax = 57% vs. 33%, sham vs. CHF operated rats), but not in the aorta. Competitive RT-PCR also revealed decreased P2X1-receptor mRNA levels in CHF operated rats in the mesenteric artery (9106 x 10(3) vs. 714 x 10(3) molecules/microgram, sham vs. CHF operated rats), while it remained unaltered in the aorta. To study the P2Y-receptor induced contractile effects, the P2X1-receptors were first desensitised with alpha beta-MeATP (10(-5) M for 8 min). After P2X1-receptors desensitisation, UTP and UDP induced strong contractions in both the mesenteric artery and in the aorta, while ATP and ADP were much less effective. These contractions were not altered by CHF, indicating that vascular contraction mediated by P2Y-receptors are unaffected by CHF. CONCLUSION: CHF induces downregulation of P2X1-receptor stimulated contraction in the mesenteric artery depending on decreased mRNA synthesis for the receptor, while the P2Y-receptor activity remains unchanged. Downregulation of P2X1-receptors appears to be specific for peripheral resistance arteries. This may represent a compensatory response to enhanced peripheral sympathetic nerve activity and increased vascular resistance in CHF. (+info)
Diinosine pentaphosphate (IP5I) is a potent antagonist at recombinant rat P2X1 receptors.
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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. (+info)
Effects of diadenosine polyphosphates (Ap(n)As) and adenosine polyphospho guanosines (Ap(n)Gs) on rat mesenteric artery P2X receptor ion channels.
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Diadenosine polyphosphates (Ap(n)As, n=3 - 7) and adenosine polyphospho guanosines (Ap(n)Gs, n=3 - 6) are naturally occurring vasoconstrictor substances found in platelets. These vasoconstrictor actions are thought to be mediated through the activation of P2X receptors for ATP. The effects of Ap(n)As and Ap(n)Gs at P2X receptors on rat mesenteric arteries were determined in contraction studies and using the patch clamp technique on acutely dissociated artery smooth muscle cells. P2X(1) receptor immunoreactivity was detected in the smooth muscle layer of artery rings. The sensitivity to alpha,beta-methylene ATP and desensitizing nature of rat mesenteric artery P2X receptors correspond closely to those of recombinant P2X(1) receptors. Ap(4)A, Ap(5)A and Ap(6)A evoked concentration dependent P2X receptor inward currents which desensitized during the application of higher concentrations of agonist. The agonist order of potency was Ap(5)A> or = Ap(6)A> or = Ap(4)A >> Ap(3)A. Ap(2)A and Ap(7)A were ineffective. Similar results were obtained in contraction studies except for Ap(7)A which evoked a substantial contraction. Ap(n)Gs (n=2 - 6)(30 microM) evoked P2X receptor inward currents in mesenteric artery smooth muscle cells. Ap(n)Gs (n=4 - 6) were less effective than the corresponding Ap(n)A. This study shows that at physiologically relevant concentrations Ap(n)As and Ap(n)Gs can mediate contraction of rat mesenteric arteries through the activation of P2X(1)-like receptors. However the activity of the longer chain polyphosphates (n=6 - 7) may be overestimated in whole tissue studies due to metabolic breakdown to yield the P2X receptor agonists ATP and adenosine tetraphosphate. British Journal of Pharmacology (2000) 129, 124 - 130 (+info)
Fertility: purinergic receptors and the male contraceptive pill.
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Knockout mice lacking the P2X(1) receptor appear normal, but fail to breed. Analysis of these mutant mice clearly shows that purinergic co-transmission has a physiological role in the was deferens. These findings also raise the possibility of developing non-hormonal ways of regulating male fertility. (+info)
A natural dominant negative P2X1 receptor due to deletion of a single amino acid residue.
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The P2X1 receptor belongs to a family of oligomeric ATP-gated ion channels with intracellular N and C termini and two transmembrane segments separating a large extracellular domain. Here, we describe a naturally occurring dominant negative P2X1 mutant. This mutant lacks one leucine within a stretch of four leucine residues in its second transmembrane domain (TM2) (amino acids 351-354). Confocal microscopy revealed proper plasma membrane localization of the mutant in stably transfected HEK293 cells. Nevertheless, voltage-clamped HEK293 cells expressing mutated P2X1 channels failed to develop an ATP or ADP-induced current. Furthermore, when co-expressed with the wild type receptor in Xenopus oocytes, the mutated protein exhibited a dose-dependent dominant negative effect on the normal ATP or ADP-induced P2X1 channel activity. These data indicate that deletion of a single apolar amino acid residue at the inner border of the P2X1 TM2 generates a nonfunctional channel. The inactive and dominant negative form of the P2X1 receptor may constitute a new tool for the study of the physiological role of this channel in native cells. (+info)