G-protein-coupled receptor regulation of P2X1 receptors does not involve direct channel phosphorylation

P2X(1) 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 mG1uR1alpha (metabotropic glutamate receptor 1alpha), P2Y(1) or P2Y(2) receptors co-expressed with P2X(1) receptors in Xenopus oocytes evoked calcium-activated chloride currents (I-CiCa) and potentiated subsequent P2X(1)-receptor-mediated currents by up to 250%. The mGluR 1alpha-receptor-mediated effects were blocked by the phospholipase C inhibitor U-73122. Potentiation was mimicked by treatment with the phorbol ester PMA. P2X receptors have a conserved intracellular PKC (protein kinase Q 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-320432 and bisindolylmaleimide 1, 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 P2X(1) receptors, and radiolabelling of phosphorylated proteins in these cells demonstrated that P2X(1) receptors are basally phosphorylated and that this level of phosphorylation is unaffected by phorbol ester treatment. This demonstrates that P2X(1) regulation does not result directly from phosphorylation of the channel, but more likely by a staurosporine-sensitive phosphorylation of an accessory protein in the P2X(1) receptor complex and suggests that in vivo fine-tuning of P2X(1) receptors by GPCRs may contribute to cardiovascular control and haremostasis.