G-protein-coupled receptor regulation of P2X(1) 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 Gα(q)-coupled mGluR1α (metabotropic glutamate receptor 1α), P2Y(1) or P2Y(2) receptors co-expressed with P2X(1) receptors in Xenopus oocytes evoked calcium-activated chloride currents (I(ClCa)) and potentiated subsequent P2X(1)-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 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 haemostasis

Nerve-evoked purinergic signalling suppresses action potentials, Ca2+ flashes and contractility evoked by muscarinic receptor activation in mouse urinary bladder smooth muscle

Contraction of urinary bladder smooth muscle (UBSM) is caused by the release of ATP and ACh from parasympathetic nerves. Although both purinergic and muscarinic pathways are important to contraction, their relative contributions and signalling mechanisms are not well understood. Here, the contributions of each pathway to urinary bladder contraction and the underlying electrical and Ca2+ signalling events were examined in UBSM strips from wild type mice and mice deficient in P2X1 receptors (P2X1−/−) before and after pharmacological inhibition of purinergic and muscarinic receptors. Electrical field stimulation was used to excite parasympathetic nerves to increase action potentials, Ca2+ flash frequency, and force. Loss of P2X1 function not only eliminated action potentials and Ca2+ flashes during stimulation, but it also led to a significant increase in Ca2+ flashes following stimulation and a corresponding increase in the force transient. Block of muscarinic receptors did not affect action potentials or Ca2+ flashes during stimulation, but prevented them following stimulation. These findings indicate that nerve excitation leads to rapid engagement of smooth muscle P2X1 receptors to increase action potentials (Ca2+ flashes) during stimulation, and a delayed increase in excitability in response to muscarinic receptor activation. Together, purinergic and muscarinic stimulation shape the time course of force transients. Furthermore, this study reveals a novel inhibitory effect of P2X1 receptor activation on subsequent increases in muscarinic-driven excitability and force generation