G-protein-coupled receptor regulation of P2X1 receptors does not involve direct channel phosphorylation.
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 Galpha(q)-coupled mGluR1alpha (metabotropic glutamate receptor 1alpha), P2Y1 or P2Y2 receptors co-expressed with P2X(1) receptors in Xenopus oocytes evoked calcium-activated chloride currents (I(ClCa)) and potentiated subsequent P2X1-receptor-mediated currents by up to 250%. The mGluR1alpha-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.
Vial
Catherine
C
Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 9HN UK.
Tobin
Andrew B
AB
Evans
Richard J
RJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002412:Cations; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating; D007668:Kidney / Q000737:chemistry / Q000196:embryology / Q000201:enzymology / Q000378:metabolism; D009865:Oocytes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism; D014981:Xenopus
D002412:Cations; C546382:P2RY1 protein, human; C546385:P2RY2 protein, human; D043562:Receptors, G-Protein-Coupled; D018094:Receptors, Metabotropic Glutamate; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; C104077:metabotropic glutamate receptor type 1; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D010738:Type C Phospholipases; D002118:Calcium