P2X1 receptors for ATP are ligand-gated cation channels, which mediate smooth muscle contraction, contribute to blood clotting and are co-expressed with a range of GPCRs (G-protein-coupled receptors). Stimulation of Gαq-coupled mGluR1α (metabotropic glutamate receptor 1α), P2Y1 or P2Y2 receptors co-expressed with P2X1 receptors in Xenopus oocytes evoked calcium-activated chloride currents (IClCa) and potentiated subsequent P2X1-receptor-mediated currents by up to 250%. The mGluR1α-receptor-mediated effects were blocked by the phospholipase C inhibitor U-73122. Potentiation was mimicked by treatment with the phor-bol ester PMA. P2X receptors have a conserved intracellular PKC (protein kinase C) site; however, GPCR- and PMA-mediated potentiation was still observed with point mutants in which this site was disrupted. Similarly, the potentiation by GPCRs or PMA was unaffected by chelating the intracellular calcium rise with BAPTA/AM [bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis-(acetoxymethyl ester)] or the PKC inhibitors Ro-32-0432 and bisindolylmaleimide I, suggesting that the regulation does not involve a calcium-sensitive form of PKC. However, both GPCR and PMA potentiation were blocked by the kinase inhibitor staurosporine. Potentiation by phorbol esters was recorded in HEK-293 cells expressing P2X1 receptors, and radiolabelling of phosphorylated proteins in these cells demonstrated that P2X1 receptors are basally phosphorylated and that this level of phosphorylation is unaffected by phorbol ester treatment. This demonstrates that P2X1 regulation does not result directly from phosphorylation of the channel, but more likely by a staurosporine-sensitive phosphorylation of an accessory protein in the P2X1 receptor complex and suggests that in vivo fine-tuning of P2X1 receptors by GPCRs may contribute to cardiovascular control and haemostasis.
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Research Article|
August 10 2004
G-protein-coupled receptor regulation of P2X1 receptors does not involve direct channel phosphorylation
Catherine VIAL;
Catherine VIAL
1Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 9HN U.K.
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Andrew B. TOBIN;
Andrew B. TOBIN
1Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 9HN U.K.
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Richard J. EVANS
Richard J. EVANS
1
1Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 9HN U.K.
1To whom correspondence should be addressed (email rje6@le.ac.uk).
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Publisher: Portland Press Ltd
Received:
December 10 2003
Revision Received:
April 21 2004
Accepted:
May 14 2004
Accepted Manuscript online:
May 14 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2004
Biochem J (2004) 382 (1): 101–110.
Article history
Received:
December 10 2003
Revision Received:
April 21 2004
Accepted:
May 14 2004
Accepted Manuscript online:
May 14 2004
Citation
Catherine VIAL, Andrew B. TOBIN, Richard J. EVANS; G-protein-coupled receptor regulation of P2X1 receptors does not involve direct channel phosphorylation. Biochem J 15 August 2004; 382 (1): 101–110. doi: https://doi.org/10.1042/BJ20031910
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